Synthesis of 4-aminophthalimide and 2,4-diaminopyrimidine C-nucleosides as isosteric fluorescent DNA base substitutes

Article abstract of DOI:10.1021/jo302768f

The 4-aminophthalimide C-nucleoside 1 was designed as an isosteric DNA base surrogate, and a synthetic route to nucleoside 1 together with the 2,4-diaminopyrimidine-C-nucleoside 2 as a potential counterbase was worked out. The key steps in both synthetic

Full text of DOI:10.1021/jo302768f

Article
pubs.acs.org/joc
Synthesis of 4‑Aminophthalimide and 2,4-Diaminopyrimidine
C‑Nucleosides as Isosteric Fluorescent DNA Base Substitutes
Michael Weinberger,†,§ Falko Berndt,‡,§ Rainer Mahrwald,‡ Nikolaus P. Ernsting,*,‡
and Hans-Achim Wagenknecht*,†
†Department of Chemistry, Karlsruhe Institute of Technology, Fritz-Haber-Weg 6, Campus Sud, Geb. 30.42, 76131 Karlsruhe,
̈
Germany
‡Institute of Chemistry, Humboldt University Berlin, Brook-Taylor-Strasse 2, 12489 Berlin, Germany
S
* Supporting Information
ABSTRACT: The 4-aminophthalimide C-nucleoside 1 was designed as an isosteric DNA
base surrogate, and a synthetic route to nucleoside 1 together with the 2,4-
diaminopyrimidine-C-nucleoside 2 as a potential counterbase was worked out. The key
steps in both synthetic routes represent a stereoselective Heck-type palladium-catalyzed
cross-coupling with 2′-deoxyribofuranoside glycal followed by stereoselective reduction
with NaBH(OAc)3. The nucleoside 1 shows a solvatofluorochromic behavior and
significantly red-shifted fluorescence in solvents of high polarity and with hydrogen
bonding capabilities. Both nucleosides 1 and 2 can be further processed to the
corresponding phosphoramidite as DNA building blocks that allow incorporation of these
chromophores as artificial DNA bases by automated DNA synthesis. The combination of
the poor stacking properties of 1 and the hydrogen bonding interface at the phthalimide
functionality that does not fit to any of natural DNA bases in the counterstrand yields
destabilization of the duplex by 4−11 °C. The fluorescence of 1 in a representative double
stranded DNA is characterized by a large Stokes’ shift and a quantum yield of
approximately 12%. These are remarkable optical properties considering the very small size of the chromophore and indicate a
high potential of these nucleoside analogues for fluorescent DNA analytics and imaging.
emission occurs in the visible range (430 nm), but quenching
inside DNA is still a problem.3,12 Other promising and
interesting structures that follow the concept of best possible
isosteric DNA base replacements include ε-ethenoadenine,13
pyrrolo-fused cytosine,14 cyclized 4-N-carbamoyl-2′-deoxycyti-
dine derivatives,15 and thieno[3,4-d]-2′-deoxyuridine.16
Although some of them are commercially available as DNA
building blocks, fluorescence inside duplex DNA is accom-
panied typically by low quantum yields that limit their
applicability. Wilhelmsson et al. published artificial nucleosides
based on 1,3-diaza-2-oxophenoxazine and 1,3-diaza-2-oxophe-
nothiazine as fluorescent substitutes with the recognition
pattern typical for cytosine. The absorption is well separated
from that of DNA (365−375 nm), and emission occurs in the
visible range (455−500 nm) with good quantum yields (ca.
20%).17 Compared to purines, the size of these nucleosides,
however, is extended by at least one benzene ring that is a fused
part of the phenoxazine or phenothiazine chromophore
structure. In a more general approach, Castellano et al.
developed push−pull systems that are based on purines as a
lead structure.18 To our knowledge, however, these purine-like
nucleosides have not yet been incorporated into oligonucleo-
tides. Finally, Hirao et al. attached bithienyl groups to artificial
INTRODUCTION
■
The preparation of fluorescent DNA and RNA conjugates is
well established but still of significant interest for chemical
bioanalytics in assays and on arrays. The ways to covalently
attach fluorophores have been studied for decades.1−3 In the
meantime, imaging of live cells attracts growing attention since
biological processes can be observed in their native environ-
ment.4 In this research field, the isomorphic or isosteric
replacement of natural DNA bases by fluorophores attracts
special interest since such labeled oligonucleotides, at least in
principle, do not significantly exceed the size of natural B-form
DNA.3,5,6 This structural feature is required especially for the
investigation of DNA−protein interactions inside cells and for
DNA polymerase-assisted replication of fluorescent oligonu-
cleotides by primer extension or PCR.2,7 The most prominent
and probably longest known example is 2-aminopurine as the
fluorescent analogue of adenine6,8,9 which has been extensively
used, e.g., to study RNA conformation equilibria10 and DNA−
protein interactions.11 The major disadvantages of this
fluorescent DNA base surrogate are the excitation in the UV-
B range (near DNA absorption range at 305 nm), the low
extinction coefficient (ε = 6,000 M−1 cm−1), the emission in the
UV-A range (370 nm), and most importantly, fluorescence
quenching inside double-stranded DNA due to charge-transfer
processes. Alternatively, pteridines have an absorption that is
better separated from the DNA absorption (330−350 nm) and
Received: December 23, 2012
Published: February 5, 2013
© 2013 American Chemical Society
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base pairs yielding fluorescent base analogues for the expansion
of the genetic alphabet.19
Scheme 1. Synthetic Steps to the Phthalimide Aglycon
Precursors 8 and 9
a
4-Aminopththalimide is a well-known, highly fluorescent, and
solvatofluorochromic dye20 that has been applied in biopol-
ymers21 but only once for oligonucleotides.22,23 The dye is
excitable at the border between UV-A and the visible range
(370−410 nm) and shows fluorescence in the visible range.
The apparent Stokes’ shift is large (up to 115 nm)24 and
depends highly on the environment, namely, the solvent
polarity and the ability for H-bonding.25,26 The time evolution
of the shift (“time-resolved dynamic Stokes’ shift”) reflects the
orientational dynamics of the surrounding medium. Accord-
ingly, 4-aminophthalimides were used as solvatofluorochromic
probes in cyclodextrines,27 polymers,28 membranes, micelles
and enzymes complexes.29 With respect to nucleic acids Hocek
et al. published most recently a synthetic protocol that allows
incorporating 4-aminophthalimide as covalent base modifica-
tion in oligonucleotides using a polymerase-assisted preparation
strategy.22 Except for this publication, we are not aware of other
attempts to prepare 4-aminophthalimide−nucleic acid con-
jugates.
In principle, 4-aminophthalimide resembles the size of
natural purines and should therefore be an isosteric DNA
base replacement in oligonucleotides. Moreover, the phthali-
mide functionality should be able to form hydrogen bonds with
counterparts of pyrimidine size that are placed in the opposite
position as part of the complementary strand. A completely
new, bioorthogonal, and fluorescent DNA base pair with fully
matching H-bonding pattern can be envisioned with 2,4-
diaminopyrimidine26 as opposite base (Figure 1).
a
Key: (a) NBS or NIS, H2SO4, 60 °C, 2 h, 94% (NBS) or 97% (NIS);
(b) KMnO4, NaOH, H2O, 100 °C, o.n.; (c) CO(NH2)2, o-xylene, 160
°C, 4 h, 32% or 35%, respectively.
halogenation into the desired position. Subsequently, the
methyl group of 4 and 5 is oxidized under standard conditions
(KMnO4 under basis conditions at 100 °C) to give 6 and 7,
respectively. Finally the phthalimide functionality was built by
condensation of urea to give 8 and 9 in 32% and 35% yield,
respectively (over the last two steps).
The phthalimides 8 and 9 represent important intermediates
since they already contain the necessary functional groups in
the right substitution pattern. They bear, most importantly, a
halogen atom to apply palladium-catalyzed cross-coupling
methodology to form a glycosidic bond to the 2′-deoxyribofur-
anoside. The necessary sugar precursor for this type of
glycosidic coupling represents glycal 10 that has been applied
successfully for the C-nucleoside synthesis in the past24 and can
be synthesized in three steps from thymidine.30
The Heck-type coupling of the aminophthalimide precursors
8 and 9 to glycal 10 forms selectively the β-anomeric bond in
acceptable yields (ca. 33%, Scheme 2). The stereoselectivity of
these reactions is the result of substrate control because the
lower face of glycal 10 is shielded effectively by the sterically
demanding TBDMS group in the 3′-position. Interestingly, the
expected silylenol ether 11 was isolated only in traces. Instead,
the 3′-deprotected ketone 12 could be purified and
subsequently reduced to nucleoside 13 by NaBH(OAc)3. The
stereoselectivity of the latter reaction is controlled by the 5′-
hydroxy group that binds the boron atom and let the hydride
attack the CO bond from the top. Finally the reduction of
the nitro group of 13 by NaSH gives the 4-aminophthalimide
C-nucleoside 1 in 88% yield.
Figure 1. Base pair surrogate consisting of 4-aminophthalimide
nucleoside 1 and 2,4-diaminopyrimidine nucleoside 2; R = 2′-
deoxyribofuranoside.
In order to incorporate nucleoside 1 into oligonucleotides by
automated phosphoramidite chemistry a corresponding DNA
building block has to be prepared. This means that the
exocyclic amino group of 1 needs to be protected, and DMT
has to be attached to the 5′-position and the phosphityl amide
group to the 3′-position. With respect to the known base
instability of the phthalimide functionality, we decided to use
the phenoxyacetyl (Pac) protecting group for the exocyclic
amino function. The attempts to protect the amino group in
the 4-position of nucleoside 1 with in situ protection of the
competing 3′- and 5′-hydroxyl groups with TMS failed
completely. Alternatively, the nitro-substituted C-nucleoside
13 was protected at the 3′- and 5′-positions with TBDMS
groups (Scheme 3). Subsequently, the nitro group of 14 was
reduced by NaSH in 70% yield and the Pac protecting group
could be attached. Finally, the two TBDMS groups of 15 were
removed and the Pac-protected nucleoside 16 was obtained in
55% yields (over two steps). In the last two synthetic steps
toward the DNA building block 18 the DMT and the
phosphoramidite group were introduced by standard proce-
dures. Preliminary experiments to prepare DNA1 (Table 1) by
automated synthesis using building block 18 failed, presumably
Herein we present the synthesis of the 4-aminophthalimide
C-nucleoside 1 and the 2,4-diaminopyrimidine C-nucleoside 2
as artificial and fluorescent DNA base surrogates, their
incorporation into oligonucleotides by automated phosphor-
amidite chemistry, and the optical properties of 1 inside double-
stranded DNA with different opposing bases, including 2. In
particular, the synthesis of oligonucleotides modified with
aminophthalimide 1 is a challenge due to the hydrolytic
instability of the imide functionality in this small chromophore
which requires special oligonucleotide synthesis and workup
conditions.
RESULTS AND DISCUSSION
■
Synthesis. The synthesis of the aminophthalimide C-
nucleoside 1 (Scheme 1) started with commercially available
2-methyl-5-nitrobenzoic acid (3) that was brominated with
NBS or iodinated with NIS to the corresponding halogenated
intermediates 4 and 5 in nearly quantitative yields. The nitro
group which forms the amino group later in the synthetic
procedure plays an important role here since it directs the
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a
Scheme 2. Heck-Type Coupling of 8 or 9 to 10 and Further Steps to the Aminophthalimide-C-nucleoside 1
a
Key: (a) P(PhF5)3, Pd(OAc)2, Et3N, MeCN, 82 °C, o.n., 33%; (b) NaBH(OAc)3, HOAc/MeCN 2:1, 0 °C, 1 h, 93%; (c) NaSH, H2O, EtOH, 78
°C, 1 h, 88%.
a
Scheme 3. Syntheses of Aminophthalimide DNA Building Blocks 18 and 20
a
Key: (a) TBDMSCl, DMF, rt, o.n. 57%; (b) NaSH, H2O, EtOH, 78 °C, 1 h, 70%; (c) (i) Pac2O, pyridine, rt, o.n., (ii) Et3N·3HF, THF, rt, o.n.,
55%; (d) DMTCl, pyridine, rt, 42 h, 52%; (e) 2-cyanoethoxy-N,N-diisopropylaminophosphanyl chloride, DIPEA, DCM, rt, 6 h, 59%; (f) DMTCl,
Et3N, pyridine, 30 °C, o.n., 64%; (g) 2-cyanoethoxy-N,N-diisopropylaminophosphanyl chloride, DIPEA, DCM, rt, 4 h, 67%.
With respect to the various synthetic detours and the
increased number of synthetic steps in the procedure described
above, an alternative synthetic route to the Pac-protected
nucleoside 16 was worked out (Scheme 4). It starts with key
intermediate 9. The nitro group was reduced with NaSH in
56% yield and the resulting amino group of 21 was directly
protected by Pac yielding 22 nearly quantitatively. As described
above, this nucleoside precursor was coupled to glycal 10 by
the Heck-type reaction to obtain the ketone 24, again in low
but still acceptable yield (27%). Stereoselective reduction with
NaBH(OAc)3 finishes in good yield this synthetic shortcut to
the Pac-protected nucleoside 16.
Because of the well-known lability of the phthalimide
functionality toward bases and nucleophiles the solid-phase
methodology had to be changed to the so-called ultramild
conditions.31 The corresponding DNA building blocks are
commercially available and bear phenoxyacetyl, isopropylphe-
noxyacetyl, and acetyl protecting groups at the DNA bases
instead of benzoyl and isobutyryl in conventional phosphor-
amidites. Accordingly, the use of concd aq ammonia can be
avoided. Alternatively, deprotection is achieved with 0.05 M
Table 1. Melting Temperatures (Tm) of DNA1-X, DNA2-X,
and DNA1-2 (X = A, G, T, C): 2.5 μM Duplex in 10 mM
Na−Pi Buffer, 250 mM NaCl, pH 7.0, λ = 260 nm, 20−90
°C, Interval 0.7 C/min
a
b
c
b
duplex
Tm (°C) ΔTm (°C)
duplex
Tm (°C) ΔTm (°C)
DNA1-A
DNA1-G
DNA1-T
DNA1-C
DNA1−2
56.0
53.5
53.0
51.2
53.6
−4.3
−11.4
−9.1
DNA2-A
DNA2-G
DNA2-T
DNA2-C
55.3
52.3
55.2
53.2
−5.0
−12.6
−6.9
−9.9
−7.9
a
Sequence of DNA1: 5′-GCT-GCA-1AC-GTC-G-3′. Counter strands
are fully complementary but vary at the base opposite to 1. In
comparison to corresponding unmodified duplexes. Sequence of
b
c
DNA2: 5′-CGA-CGT-2TG-CAG-C-3′. Counter strands are fully
complementary but vary at the base opposite to 2.
due to the nucleophilicity of the phthalimide nitrogen. Hence, a
second DMT group was introduced at this position of 16 and
subsequent phosphitylation of 19 gave the fully protected DNA
building block 20.
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a
Scheme 4. Shorter Alternative Route to the Pac-Protected Aminophthalimide Nucleoside 16
a
Key: (a) NaSH, H2O, EtOH, 78 °C, 1 h, 56%; (b) PacCl, imidazole, THF, pyridine, rt, 4 h, 95%; (c) 10, P(PhF5)3, Pd(OAc)2, Et3N, MeCN, 82 °C,
o.n., 27%; (d) NaBH(OAc)3, HOAc/MeCN 2:1, 0 °C, 1 h, 94%.
K2CO3 in MeOH, typically within 4 h at rt. The first attempts
to deprotect DNA1 that was synthesized automatically using
the phosphoramidite 20 were not successful. Mass spectrom-
etry revealed that the protecting groups, especially the Pac
groups at the adenines, could be cleaved off; however, the Pac
group at the 4-aminophthalimide could not. Hence, the
deprotection conditions were varied. This included additionally
Cs2CO3 and K-t-BuO, each in MeOH or i-PrOH, NH3 in H2O,
MeNH2 in EtOH (1:1), and NaOH in MeOH (4:1). Several
temperatures from 30 to 80 °C and incubation times from 2 to
24 h were tried. These experiments revealed that only the use
of 0.05 M K2CO3 in MeOH, and an incubation time of 48 h at
65 °C gave successfully the completely deprotected oligonu-
cleotide DNA1.
quantitative yields. Subsequent substitution of chlorine in 28
by ammonia yielded 2 in 73% yield.
With the unprotected diaminopyrimidine nucleoside 2 in
hand, we tried to synthesize the corresponding phosphor-
amidite for later oligonucleotide preparation. Accordingly, the
exocyclic amino functions were protected as phenoxyaceta-
mides and the primary 5′-hydroxy-function by DMT group.
The phenoxyacetamide group was selected to accomplish a
faster cleavage by ammonia under milder conditions compared
to benzamide. A first one-pot approach, in situ TMS protection
of the hydroxy groups and subsequent introduction of the
phenoxyacetyl moiety, led to an inseparable mixture of diverse
protected nucleosides in low yields.
These results forced us to use another synthetic route to the
protected phosphoramidite. Starting with the unprotected 2,4-
dichloropyrimidine nucleoside 28, the direct amination should
be realized by introduction of benzamides33 via a palladium-
catalyzed Buchwald−Hartwig reaction. In a first series, we
tested 2,4-dichloropyrimidine 29 as model substrate to find the
optimal reaction conditions (working catalyst, etc.). Using a
combination of Pd(dba)2 and Xantphos as catalyst, 2,4-
bis(benzoylamino)pyrimidine 30 was isolated in 78% yield
(Scheme 6).
For the synthesis of the 2,4-diaminopyrimidine C-nucleoside
2 a synthetic route developed by Kubelka et al.32 was adopted
(Scheme 5). To this end 3-TBDMS-protected glycal 10 was
Scheme 5. Synthetic Route to 2,4-Diaminopyrimidine-C-
a
nucleoside 2 Developed by Kubelka32
Scheme 6. Buchwald−Hartwig Amination of Model
Substrate 2,4-Dichloropyrimidine 29
Upon transfer of these conditions to the unprotected 2,4-
dichloropyrimidine nucleoside 28, a mixture of starting
material, monosubstituted product, and monosubstituted
pyrimidine nucleoside in its pyranoside form was obtained.
The low conversion might be due to the low solubility of 28 in
toluene. We therefore turned to the more polar 1,4-dioxane and
increased the amount of catalyst. By increasing the temperature
to 100 °C, we were able to force the full substitution. At these
elevated temperatures the formation of the pyranoside form is
very likely, so we had to block the 5′-hydroxy group. The
protection as DMT-ether seemed to be obvious, but the DMT-
group is not stable under amination conditions.
a
Key: (a) Pd(OAc)2, P(PhF5)3, Ag2CO3, CHCl3, 12 h, 70 °C; (b)
AcOH, TBAF, THF, 1 h, 0 °C, 34% overall yield; (c) NaBH(OAc)3,
AcOH, CH3CN, 30 min, 0 °C, 92%; (d) NH3, MeOH, 12 h, 120 °C,
73%.
reacted with 2,4-dichloro-5-iodopyrimidine 25 in the presence
of palladium acetate and tris(pentafluorophenyl)phosphine.
Since the silylenol ether 26 already starts to hydrolyze to 3′-
ketoriboside 27 under these reaction conditions, the crude
silylenol ether was directly converted to 27 by addition of acetic
acid and TBAF. The β ketoriboside 27 was isolated in 34%
overall yield with high stereoselectivity. The α-anomer could
not be detected via TLC nor NMR. Stereoselective reduction of
the ketone 27 by NaBH(OAc)3 proceeded in nearly
As a result of this optimization work the following protocol
was elaborated (Scheme 7). Initial protection of the 5′-hydroxy
group of 28 was accomplished with TBDMSCl and imidazole
in 85% yield. Subsequent Buchwald−Hartwig amination of 31
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a
Scheme 7. Synthesis of 2,4-Bis(benzylamino)pyrimidine Nucleoside 33 and the Corresponding Protected Phosphoramidite 35
a
Key: (a) TBDMSCl, imidazole, DMF, 16 h, rt, 85%; (b) BzNH2, Pd2(dba)3, Xantphos, CsCO3, dioxane, 10 h, 100 °C, 82%; (c) NEt3·3HF, THF, 2
h, rt, 70%; )d) DMTCl, pyridine, 2 d, rt, 86%; (e) 2-cyanoethoxy-N,N-diisopropylaminophosphanyl chloride, DIPEA, CH3CN, 2 h, rt, 60%.
Figure 2. Solvatochromism (left) and solvatofluorochromism (right) of aminophthalimide nucleoside 1. λexc = 385 nm.
Figure 3. UV/vis absorption and fluorescence of modified single-stranded DNA1, double-stranded DNA1A−DNA1C and DNA1-2. 2.5 μM with
250 mM NaCl in 10 mM sodium phosphate buffer pH = 7.0, λexc = 385 nm.
with benzamide gave TBDMS-protected 2,4-bis(benzoyl-
amino)pyrimidine nucleoside 32 in 82% yield. The TBDMS-
group of 32 was removed by NEt3·3HF in THF in 70% yield.
The bis(benzoylamino)pyrimidine nucleoside 33 was then
protected with DMTCl at the 5′-hydroxy group and finally
converted to phosporamidite 34 by 2-cyanoethoxy-N,N-
diisopropylaminophosphanyl chloride. The DNA building
block 35 was subjected to automated oligonucleotide synthesis.
DNA2 was deprotected and removed from the CPG by
aqueous ammonia and subsequently purified by preparative
HPLC.
Steady-State Optical Spectroscopy. The aminophthali-
mide C-nucleoside 1 was characterized both as monomer and
as modification in DNA1 by UV/vis absorption and steady-
state fluorescence spectroscopy (Figure 2). The isolated
nucleoside exhibits a solvatochromic behavior that is similar
to the chromophore alone, including a very pronounced red-
shift of absorption and emission with increasing solvent
polarity. The fluorescence is additionally red-shifted in solvents
with hydrogen-bonding capabilities. The quantum yield was
determined to be 0.033 which was higher than the literature
value of 0.022.20 Overall, the sugar modification has only minor
influence on the optical properties of 4-aminophthalimide in C-
nucleoside 1.
DNA1 was hybridized with five different complementary
oligonucleotides as counter strands to five different double-
stranded samples. The only difference represents the base (A,
G, T, C, 2) that was opposite to the aminophthalimide DNA
base surrogate (Table 1). The determination of the melting
temperatures of DNA1-X (X = A, G, T, C) revealed strong
destabilization compared to the corresponding nonmodified
duplexes. It became clear that the combination of the poor
stacking properties of the 4-aminophthalimide chromophore as
artificial DNA base and the hydrogen bonding interface at the
phthalimide functionality that does not fit to any of natural
DNA bases as counter bases yields the observed effect. Similar
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destabilizing effects were obtained with DNA2-X (X = A, G, T,
C) again varying in the base that is opposite to the 2,4-
diaminopyrimidine DNA base surrogate. Finally DNA1 and
DNA2 were hybridized to duplex DNA1-2. Since they are fully
complementary to each other the aminophthalimide 1 is placed
opposite to the 2,4-diaminopyrimidine 2. We assume that these
two base surrogates form a base pair by their complementary
hydrogen-bonding interface which is orthogonal to the natural
base pairs. The Tm measurements do not reflect this base pair
although it should be mentioned that the destabilizing effect is
not doubled due to the presence of two different modifications
in DNA1-2. NMR studies are currently performed to support
this interpretation.
The UV/vis absorption spectra of single-stranded DNA1,
double-stranded DNA1-X (X = A, G, T, C), and DNA1-2 show
the presence of the aminophthalimide chromophore by an
additional absorption band between 350 and 420 nm that is
well separated from the absorption range of the nucleic acid
and allows selective excitation (Figure 3). When excited at 385
nm, fluorescence of single stranded DNA1 shows a maximum
at approximately 535 nm which shifts to 525 nm upon
hybridization with the counter strands. Hence, the interior of
the DNA base stack resembles the polarity and properties of
MeOH. The Stokes’ shift is remarkably large (140 nm). Single-
stranded DNA1 exhibits a quantum yield of 0.060 which
represents a ca. 1.8 fold increase compared to the nucleoside
monomer. The fluorescence is further increased to at least
approximately double intensity in case of DNA1-A, DNA1-T,
DNA1-C and DNA1-2 but not DNA1-G. DNA1-2 exhibits a
quantum yield of 0.114. Time-resolved fluorescence studies will
reveal the details of the photophysical interactions of 4-
aminophthalimide in DNA.
The fluorescence of 1 in double-stranded DNA is
characterized by a large Stokes’ shift (excitation at 385 nm,
emission at 525 nm) and a quantum yield of approximately
12%. These are remarkable optical properties considering the
very small size of the chromophore. The 4-aminophthalimide
nucleoside is an isosteric surrogate for purine nucleosides in
DNA. The small steric demand of this fluorescent nucleoside
represents an ideal prerequisite that 4-aminophthalimide-
modified DNA does not significantly exceed the size of natural
B-form duplexes. This structural feature is especially interesting
for the investigation of DNA−protein interactions inside cells.
Additionally it should allow the DNA polymerase-assisted
replication of fluorescent oligonucleotides by primer extension
or PCR. Moreover, together with 2,4-diaminopyrimidine as
counter base a completely new, bioorthogonal and fluorescent
DNA base pair will be created for chemical biology with nucleic
acids, especially with high potential for live cell imaging.
EXPERIMENTAL SECTION
■
General Experimental Methods. Chemicals and dry solvents
were purchased from commercial suppliers and were used without
further purification unless otherwise mentioned. TLC was performed
on silica gel F254 coated aluminum foil. Flash chromatography was
carried out with silica gel (43−60 μm). Optical−spectroscopic
measurements were recorded in Na−Pi buffer solution (10 mM, pH
7) or in the mentioned solvents using quartz glass cuvettes (l = 10
mm). Mass spectra were measured in the central analytical facility of
the institute. HRMS were measured using ESI with TOF analyzer
(marked with ESI-TOF) or ESI with FT-ICR analyzer (marked with
ESI-FT-ICR). NMR spectra were recorded on a 300 or 600 MHz
spectrometer at 300 K in deuterated solvents. Chemical shifts are given
in ppm relative to TMS. Complete assignment of all NMR signals was
performed using a combination of 2D-NMR (H,H−COSY, H,C-
HSQC, H,C-HMBC, and NOESY) experiments. Absorption spectra
and melting temperatures (2.5 μM DNA, 20−90 °C, 0.7 °C/min, step
width 0.5 °C) were recorded on a UV/vis spectrometer equipped with
a 6 × 6 cell changer unit. Fluorescence was measured on a fluorimeter
with a step width of 1 nm an integration time of 0.2 s. All spectra were
recorded with an excitation and emission bypass of 5 nm and are
corrected for Raman emission of the buffer solution
CONCLUSIONS
■
A synthetic route to the 4-aminophthalimide C-nucleoside 1
and its incorporation into an oligonucleotide has been
successfully worked out. The key steps represent a stereo-
selective Heck-type palladium-catalyzed cross-coupling of the
halogenated precursors 8, 9, or 22 of the 4-aminophthalimide
chromophore and glycal 10 followed by stereoselective
reduction with NaBH(OAc)3. The nucleoside 1 shows a
solvatofluorochromic behavior and significantly red-shifted
fluorescence in solvents of high polarity and with hydrogen
bonding capabilities. Nucleoside 1 can be further processed to
the corresponding phosphoramidite as DNA building block
that allows incorporating the aminophthalimide as an artificial
DNA base by automated DNA synthesis. It turned out that
only the combination of a Pac-protecting group at the 4-amino
function and a DMT group at the phthalimide function of
phosphoramidite 20, the so-called ultramild conditions for
oligonucleotide synthesis and perfectly tuned deprotection
conditions yielded the 4-aminophthalimide-modifided oligonu-
cleotide DNA1.
3-Bromo-2-methyl-5-nitrobenzoic Acid (4). 2-Methyl-5-nitro-
benzoic acid (3, 2.00 g, 11.0 mmol) was dissolved in concd sulfuric
acid (10 mL) and heated to 60 °C. N-Bromosuccinimide (2.36 g, 13.3
mmol) was added in three portions over 30 min. The reaction mixture
was stirred at 60 °C for an additional 2 h and then poured onto ice (24
g). The precipitate was filtered off and washed with cold water and
1
hexane affording 4 (2.70 g, 10.4 mmol, 94%) as a white solid: H
NMR (300 MHz, DMSO-d6) δ 8.53 (d, J = 2.5 Hz, 1H, H-4), 8.44 (d,
J = 2.5 Hz, 1H, H-6), 2.65 (s, 3H, CH3); 13C NMR (75 MHz, DMSO-
d6) δ 166.7 (COOH), 145.6, 145.1, 134.4, 129.2 (C-4), 126.6, 123.4
(C-6), 20.8 (CH3); HRMS (ESI-TOF) m/z calcd for C8H5BrNO4
[M−H]− 257.9407, found 257.9398.
3-Iodo-2-methyl-5-nitrobenzoic Acid (5). 2-Methyl-5-nitro-
benzoic acid (3, 2.02 g, 11.1 mmol) was dissolved in concd sulfuric
acid (10 mL) and heated to 60 °C. N-Iodosuccinimide (2.93 g, 13.0
mmol) was added in three portions over 30 min. The reaction mixture
was stirred at 60 °C for an additional 2 h and then poured onto ice (24
g). The precipitate was filtered off and washed with cold water and
hexane to afford 5 (3.31 g, 10.8 mmol, 97%) as a white solid: 1H NMR
(300 MHz, DMSO-d6) δ 8.68 (d, J = 2.5 Hz, 1H, H-4), 8.43 (d, J = 2.5
Hz, 1H, H-6), 2.67 (s, 3H, CH3); 13C NMR (75 MHz, DMSO-d6) δ
166.9 (COOH), 148.1, 145.3, 135.3 (C-4), 133.2, 123.9 (C-6), 104.4,
26.3 (CH3); HRMS (ESI-TOF) m/z calcd for C8H5INO4 [M−H]−
305.9269, found 305.9256.
The 2,4-diaminopyrimidine-C-nucleoside 2 was obtained by
palladium-catalyzed Heck-type coupling of 2,4-dichloro-5-
iodopyrimidine 25 with glycal 10 and subsequent aminolysis
of the chlorine atoms. The protected 2,4-bis(benzylamino)-
pyrimidine-C-nucleoside phosphoramidite 35 was synthesized
by Buchwald−Hartwig amination of 31 with benzamide. The
DNA building block 35 was successfully incorporated into
DNA by automated phosphoramidite synthesis yielding DNA2,
the completely matched counter strand to DNA1.
3-Brom-5-nitrophthalimide (8). Compound 4 (2.60 g, 10.0
mmol) was dissolved in water (78 mL) and 2 M NaOH solution (5.2
mL) and heated to 60 °C. Potassium permanganate (6.32 g, 40.0
mmol) was added, and the reaction was stirred under reflux overnight.
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The reaction mixture was filtered using Celite and washed with hot
water. The solution was brought to pH 1 with hydrochloric acid and
the solvent was evaporated to dryness. The residue containing 6 was
taken up in o-xylene (116 mL), urea (1.80 g, 30.0 mmol) was added,
and the reaction mixture was stirred under reflux for 4 h. The solution
was filtered and the solvent was removed under reduced pressure. The
residue was purified by flash chromatography on silica gel (CH2Cl2/
EtOAc = 30:1) to obtain 8 (864 mg, 3.19 mmol, 32%) as a yellow
1H, NH), 8.93 (d, J = 2.0 Hz, 1H, H-4), 8.38 (d, J = 2.0 Hz, 1H, H-6),
6.04 (dd, J = 10.3, 6.4 Hz, 1H, H-1′), 5.16 (t, J = 5.3 Hz, 1H, OH-5′),
4.19 (t, J = 3.0 Hz, 1H, H-4′), 3.83−3.70 (m, 2H, H-5′), 3.04 (dd, J =
18.0, 6.5 Hz, 1H, H-2′), 2.34 (dd, J = 18.0, 10.3 Hz, 1H, H-2′); 13C
NMR (126 MHz, DMSO-d6) δ 213.0 (C-3′), 167.9 (CO), 167.1
(CO), 151.7, 142.3, 134.4, 132.8, 126.0 (C-4), 116.6 (C-6), 82.6
(C-4′), 71.2 (C-1′), 60.4 (C-5′), 44.2 (C-2′); HRMS (ESI-TOF) m/z
calcd for C13H9N2O7 [M−H]− 305.0415, found 305.0414.
1
solid: Rf 0.75(CH2Cl2/EtOAc = 5:1); H NMR (400 MHz, DMSO-
1-(5-Nitrophthalimid-3-yl)-1,2-dideoxy-β-D-ribofuranose
(13). In a degassed mixture of acetic acid and acetonitrile (2:1, 5 mL),
12 (80 mg, 0.26 mmol) was dissolved under Ar atmosphere at 0 °C.
Sodium triacetoxyborohydride (83 mg, 0.39 mmol) was added, and
the reaction mixture was stirred at 0 °C for 1 h. A mixture of water and
ethanol (1:1, 3.3 mL) was added, and the solvents were removed to
dryness. The residue was purified by flash chromatography on silica gel
(CHCl3/MeOH 1:0 to 4:1) yielding 13 (75 mg, 0.24 mmol, 93%) as a
d6) δ 11.97 (s, 1H, NH), 8.73 (d, J = 1.8 Hz, 1H, H-4), 8.39 (d, J = 1.8
Hz, 1H, H-6); 13C NMR (101 MHz, DMSO-d6) δ 166.0 (CO),
165.8 (CO), 151.3, 136.0, 134.7, 133.2 (C-4), 117.7, 116.8 (C-6);
HRMS (ESI-TOF) m/z calcd for C8H2BrN2O4 [M−H]− 268.9203,
found 268.9215.
3-Iodo-5-nitrophthalimide (9). Compound 5 (2.80 g, 9.12
mmol) was dissolved in water (84 mL) and 2 M NaOH solution (5.6
mL) and heated to 60 °C. Potassium permanganate (5.78 g, 36.6
mmol) was added, and the reaction was stirred under reflux overnight.
The reaction mixture was filtered using Celite and washed with hot
water. The solution was brought to pH 1 with hydrochloric acid, and
the solvent was evaporated to dryness. The residue containing 7 was
taken up in o-xylene (123 mL), urea (1.65 g, 27.5 mmol) was added,
and the reaction mixture was stirred under reflux for 4 h. The solution
was filtered and the solvent was removed under reduced pressure. The
residue was purified by flash chromatography on silica gel (CH2Cl2/
EtOAc = 30:1) to obtain 9 (1.02 g, 3.21 mmol, 35%) as a yellow solid:
1
white solid: Rf 0.24 (CHCl3/MeOH = 9:1); H NMR (500 MHz,
DMSO-d6) δ 11.80 (s, 1H, NH), 8.75 (dd, J = 1.9, 0.7 Hz, 1H, H-4),
8.30 (d, J = 2.0 Hz, 1H, H-6), 5.84 (dd, J = 9.7, 6.0 Hz, 1H, H-1′), 5.18
(d, J = 3.9 Hz, 1H, OH-3′), 4.91 (t, J = 5.3 Hz, 1H, OH-5′), 4.25−4.19
(m, 1H, H-3′), 3.90 (td, J = 4.7, 2.5 Hz, 1H, H-4′), 3.57 (td, J = 4.9,
1.9 Hz, 1H, H-5′), 2.37 (ddd, J = 12.8, 6.1, 2.1 Hz, 1H, H-2′), 1.77
(ddd, J = 12.7, 9.8, 5.7 Hz, 1H, H-2′); 13C NMR (126 MHz, DMSO-
d6) δ 168.0 (CO), 167.2 (CO), 151.5, 144.8, 134.5, 132.3, 125.7
(C-4), 116.0 (C-6), 88.1 (C-4′), 74.2 (C-1′), 72.1 (C-3′), 62.0 (C-5′),
42.7 (C-2′); HRMS (ESI-TOF) m/z calcd for C13H11N2O7 [M−H]−
307.0572, found 307.0570.
1
Rf 0.76 (CH2Cl2/EtOAc = 5:1); H NMR (300 MHz, DMSO-d6) δ
11.94 (s, 1H, NH), 8.86 (d, J = 1.9 Hz, 1H, H-4), 8.39 (d, J = 1.9 Hz,
1H, H-6); 13C NMR (75 MHz, DMSO-d6) δ 166.9 (CO), 165.4
(CO), 150.7, 139.0 (C-4), 137.7, 135.2, 117.1 (C-6), 90.3; HRMS
(ESI-TOF) m/z calcd for C8H2IN2O4 [M−H]− 316.9065, found
316.9084.
5-Amino-3-iodophthalimide (21). A solution of 9 (1.03 g, 3.29
mmol) in ethanol (40 mL) was brought to boiling, and an aqueous
sodium hydrogen sulfide solution (1.82 M, 3.0 mL, 5.46 mmol) was
added dropwise. The reaction mixture was stirred under reflux for 1 h.
The solvent was removed to dryness, and the residue was purified by
flash chromatography on silica gel (EtOAc) to afford 21 (522 mg, 1.82
mmol, 56%) as a light yellow solid: Rf 0.27 (CH2Cl2/acetone = 9:1);
1H NMR (400 MHz, DMSO-d6) δ 10.92 (s, 1H, NH), 7.21 (d, J = 2.0
Hz, 1H), 6.87 (d, J = 2.0 Hz, 1H), 6.55 (s, 2H, NH2); 13C NMR (101
MHz, DMSO-d6) δ 168.0 (CO), 167.4 (CO), 155.0, 136.6, 126.4
(C-4), 117.9, 107.1 (C-6), 90.8; HR-MS (ESI-TOF) m/z calcd for
C8H6IN2O2 [M + H]+ 288.9468, found 288.9490.
1-(5-Aminophthalimid-3-yl)-1,2-dideoxy-β-D-ribofuranose
(1). A solution of 13 (110 mg, 0.36 mmol) in ethanol (5.5 mL) was
brought to boiling, and an aqueous sodium hydrogen sulfide solution
(1.82 M, 330 μL, 0.60 mmol) was added dropwise. The reaction
mixture was stirred under reflux for 1 h. The solvent was removed to
dryness, and the residue was purified by flash chromatography on silica
gel (CHCl3/EtOH 1:0 to 3:2) to afford 1 (87 mg, 0.31 mmol, 88%) as
1
a light yellow solid: Rf 0.19 (CHCl3/EtOH = 5:1); H NMR (500
MHz, DMSO-d6) δ 10.66 (s, 1H Imid-NH), 6.97 (d, J = 2.0 Hz, 1H,
H-4), 6.74 (d, J = 2.0 Hz, 1H, H-6), 6.38 (s, 2H, NH2), 5.60 (dd, J =
10.0, 5.8 Hz, 1H, H-1′), 5.07 (d, J = 3.9 Hz, 1H, OH-3′), 4.75 (t, J =
5.6 Hz, 1H, OH-5′), 4.17−4.10 (m, 1H, H-3′), 3.80 (ddd, J = 6.2, 5.2,
2.5 Hz, 1H, H-4′), 3.54 (dt, J = 10.8, 5.3 Hz, 1H, H-5′), 3.41 (dt, J =
11.5, 6.0 Hz, 1H, H-5′), 2.30 (ddd, J = 12.7, 5.9, 1.8 Hz, 1H, H-1′),
1.60 (ddd, J = 12.7, 10.0, 5.8 Hz, 1H, H1′); 13C NMR (126 MHz,
DMSO-d6) δ (ppm) = 169.5 (CO), 169.3 (CO), 154.8, 144.5,
135.8, 113.2, 112.6 (C-4), 105.6 (C-6), 87.6 (C-4′), 74.7 (C-1′), 72.3
(C-3′), 62.5 (C-5′), 42.4 (C-2′); HRMS (ESI-TOF) m/z calcd for
C13H15N2O5 [M + H]+ 279.0975, found 279.1000.
5-(N-Phenoxyacetylamino)-3-iodophthalimide (22). Under Ar
atmosphere, 21 (300 mg, 1.04 mmol) and imidazole (510 mg, 7.50
mmol were dissolved in anhyd THF (25 mL) and anhyd pyridine (10
mL). Phenoxyacetyl chloride (865 μL, 6.25 mmol) was added
dropwise, and the reaction mixture was stirred for 4 h at rt. Ethanol
(1 mL) was added, and the solvents were removed under reduced
pressure. The residue was purified by flash chromatography on silica
gel (CH2Cl2/acetone 1:0 to 15:1) to afford 22 (417 mg, 0.99 mmol,
95%) as a white solid: Rf 0.55(CH2Cl2/acetone = 9:1); 1H NMR (500
MHz, DMSO-d6) δ 11.42 (s, 1H, Imid-NH), 10.66 (s, 1H, Amid-NH),
8.45 (d, J = 1.8 Hz, 1H, H-4), 8.15 (d, J = 1.7 Hz, 1H, H-6), 7.35−7.30
1-(5-Nitrophthalimid-3-yl)-3,5-di-O-tert-butyldimethylsilyl-
1,2-dideoxy-β-D-ribofuranose (14). Under Ar atmosphere, 13 (158
mg, 0.51 mmol) and imidazole (162 mg, 1.07 mmol) were dissolved in
anhyd DMF (3.5 mL), and TBDMS-Cl (162 mg, 1.07 mmol) was
added. The reaction mixture was stirred at rt overnight. Water (3.5
mL) was added, and the solution was extracted with hexane (3 × 10
mL). The combined organic layers were dried (Na2SO4), and the
solvent was removed under reduced pressure yielding 14 (158 mg,
0.29 mmol, 57%) as a white solid: Rf 0.6 (CH2Cl2/EtOAc = 15:1); 1H
NMR (300 MHz, DMSO-d6) δ 11.79 (s, 1H, NH), 8.63 (dd, J = 2.0,
0.8 Hz, 1H, H-4), 8.32 (d, J = 2.0 Hz, 1H, H-6), 5.86 (dd, J = 9.9, 5.7
Hz, 1H, H-1′), 4.43−4.34 (m, 1H, H-3′), 3.99−3.90 (m, 1H, H-4′),
3.80−3.74 (m, 2H, H-5′), 2.40 (ddd, J = 12.7, 5.7, 1.6 Hz, 1H, H-2′),
1.82 (ddd, J = 12.5, 10.0, 5.5 Hz, 1H, H-2′), 0.91 [s, 9H, C(CH3)3],
(m, 2H, Aryl-H), 7.04−6.95 (m, 3H, Aryl-H), 4.77 (s, 2H, CH2); 13
C
NMR (126 MHz, DMSO-d6) δ 167.8 (CO), 167.7 (CO), 166.9
(CO), 157.6, 143.8, 135.4, 133.6, 129.6 (C-4), 127.3, 121.4, 114.7
(C-6), 113.1, 89.9, 67.1 (CH2); HR-MS (ESI-TOF) m/z calcd for
C16H12IN2O4 [M + H]+ 422.9836, found 422.9867.
0.85 [s, 9H, C(CH3)3], 0.11 (s, 3H, CH3), 0.09 (s, 9H, 3 × CH3); 13
C
1-(5-Nitrophthalimid-3-yl)-1,2,3-trideoxy-3-oxo-β-D-ribofur-
anose (12). Under Ar atmosphere, 10 (325 mg, 1.41 mmol) was
dissolved in dry acetonitrile (29 mL). Compound 9 (408 mg, 1.28
mmol), tris(pentafluorophenyl)phosphine (273 mg, 0.51 mmol),
palladium(II) acetate (60 mg, 0.27 mmol), and Et3N (355 μL, 2.56
mmol) were added, and the reaction was stirred under reflux
overnight. The solvent was removed to dryness, and the residue was
purified by flash chromatography on silica gel (CH2Cl2/acetone = 9:1)
to obtain 12 (128 mg, 0.42 mmol, 33%) as a white solid: Rf 0.31
NMR (126 MHz, DMSO-d6) δ 167.9 (CO), 167.1 (CO), 151.5,
144.1, 134.5, 132.3, 125.7 (C-4), 116.0 (C-6), 88.1 (C-4′), 74.2 (C-
1′), 72.1 (C-3′), 62.0 (C-5′), 42.7 (C-2′), 25.8 [C(CH3)3], 25.7
[C(CH3)3], 17.8 [C(CH3)3], 17.8 [C(CH3)3], −3.2 (CH3), −4.8
(CH3); HRMS (ESI-TOF) m/z calcd for C25H41N2O7Si2 [M + H]+
537.2447, found 537.2477.
1-(5-Aminophthalimid-3-yl)-3,5-di-O-tert-butyldimethylsilyl-
1,2-dideoxy-β-D-ribofuranose (15). A solution of 14 (401 mg, 0.75
mmol) in ethanol (11.5 mL) was brought to boiling, and an aqueous
1
(hexane/EtOAc = 1:1); H NMR (500 MHz, DMSO-d6) δ 11.87 (s,
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sodium hydrogen sulfide solution (1.82 M, 690 μL, 1.26 mmol) was
added dropwise. The reaction mixture was stirred under reflux for 1 h.
The solvent was removed to dryness, and the residue was purified by
flash chromatography on silica gel (CH2Cl2/EtOAc 1:0 to 9:1) to
afford 15 (264 mg, 0.52 mmol, 70%) as a light yellow solid: Rf 0.14
(CH2Cl2/EtOAc = 15:1); 1H NMR (300 MHz, DMSO-d6) δ 10.65 (s,
1H, Imid-NH), 6.89 (d, J = 1.9 Hz, 1H, H-4), 6.74 (d, J = 2.0 Hz, 1H,
H-6), 6.39 (s, 2H, NH2), 5.60 (dd, J = 10.3, 5.2 Hz, 1H, H-1′), 4.32 (d,
J = 4.8 Hz, 1H, H-3′), 3.90−3.79 (m, 1H, H-4′), 3.73 (dd, J = 10.5, 4.5
Hz, 1H, H-5′), 3.49 (dd, J = 10.4, 7.5 Hz, 1H, H-5′), 2.35 (dd, J =
12.8, 5.2 Hz, 1H, H-2′), 1.65 (ddd, J = 12.6, 10.5, 5.3 Hz, 1H, H-2′),
0.90 [s, 9H, C(CH3)3], 0.87 [s, 9H, C(CH3)3], 0.10 (s, 3H, CH3),
0.09−0.07 (m, 9H, 3x CH3); 13C NMR (126 MHz, DMSO-d6) δ 169.5
(CO), 169.2 (CO), 154.9, 143.6, 135.9, 113.2, 112.2 (C-4), 105.6
(C-6), 87.5 (C-4′), 75.1 (C-1′), 74.5 (C-3′), 63.6 (C-5′), 41.9 (C-2′),
25.8 [C(CH3)3], 25.7 [C(CH3)3], 18.0 [C(CH3)3], 17.8 [C(CH3)3],
−4.7 (CH3), −4.8 (CH3), −5.35 (CH3), −5.39 (CH3); HRMS (ESI-
TOF) m/z calcd for C25H43N2O5Si2 [M + H]+ 507.2705, found
507.2718.
(126 MHz, DMSO-d6) δ 169.0 (CO), 168.8 (CO), 167.6 (C
O), 157.7, 143.8, 134.4, 129.6 (Aryl-CH), 122.1, 121.3 (Aryl-CH),
120.2 (C-4), 114.6 (Aryl-CH), 111.8 (C-6), 87.8 (C-4′), 74.4 (C-1′),
72.4 (C-3′), 67.0 (CH2), 62.5 (C-5′), 42.4 (C-2′); HRMS (ESI-TOF)
m/z calcd for C21H21N2O7 [M + H]+ 413.1343, found 413.1364.
1-(5-(N-Phenoxyacetyl)amino-N-(dimethoxytrityl)-
phthalimid-3-yl)-5-O-(dimethoxytrityl)-1,2-dideoxy-β-D-ribo-
furanose (19). Under Ar atmosphere, 16 (118 mg, 0.29 mmol) and
DMT-Cl (291 mg, 0.86 mmol) were dissolved in anhyd pyridine, and
Et3N (139 μL, 1.00 mmol) was added. The reaction mixture was
stirred at 30 °C overnight. The solvent was removed under reduced
pressure, and the residue was purified by flash chromatography on
silica gel (hexane/EtOAc = 2:3, + 0.1% Et3N) yielding 19 (187 mg,
0.18 mmol, 64%) as a white solid: Rf 0.48 (hexane/EtOAc = 1:1); 1H
NMR (500 MHz, DMSO-d6) δ 10.60 (s, 1H, Amid-NH), 8.06 (d, J =
1.7 Hz, 1H, H-4), 7.97 (d, J = 1.8 Hz, 1H, H-6), 7.41−7.34 (m, 7H,
Aryl-H), 7.31−7.24 (m, 8H, Aryl-H), 7.23−7.17 (m, 4H, Aryl-H),
7.15−7.10 (m, 2H, Aryl-H), 6.99−6.95 (m, 3H, Aryl-H), 6.88 (d, J =
8.8 Hz, 3H, Aryl-H), 6.82 (d, J = 8.9 Hz, 4H, Aryl-H), 5.61 (dd, J =
9.7, 5.8 Hz, 1H, H-1′), 5.18 (d, J = 4.1 Hz, 1H, OH-3′), 4.70 (s, 2H,
CH2), 4.10−4.04 (m, 1H, H-3′), 3.94 (td, J = 5.5, 2.8 Hz, 1H, H-4′),
3.73−3.70 (2 × s, 12H, OCH3), 3.25 (dd, J = 9.9, 5.4 Hz, 1H, H-5′),
3.04 (dd, J = 9.8, 5.7 Hz, 1H, H-5′), 2.26 (ddd, J = 12.1, 5.7, 1.5 Hz,
1H, H-2′), 1.58 (ddd, J = 12.6, 10.0, 5.9 Hz, 1H, H-2′); 13C NMR
(126 MHz, DMSO-d6) δ 167.3 (CO), 167.2 (CO), 167.1 (C
O), 158.0, 157.6, 157.4, 144.9, 143.8, 142.9, 135.6, 135.0, 133.3, 129.7,
129.6, 129.5, 127.8, 127.7, 127.4, 126.6, 125.7, 121.3 (C-4), 114.6,
113.2, 112.8 (C-6), 85.8 (C-4′), 85.5, 74.5 (C-1′), 72.6 (C-3′), 72.0,
70.0 (CH2), 64.1 (C-5′), 55.0 (OCH3), 54.9 (OCH3), 42.2 (C-2′);
HRMS (ESI-TOF) m/z calcd for C63H55N2O11 [M − H]− 1015.3811,
found 1015.3820.
1-(5-(N-Phenoxyacetyl)aminophthalimid-3-yl)-1,2,3-tri-
deoxy-3-oxo-β-D-ribofuranose (24). Under Ar atmosphere, 22
(735 mg, 1.74 mmol) was dissolved in dry acetonitrile (22 mL).
Compound 10 (441 mg, 1.91 mmol), tris(pentafluorophenyl)-
phosphine (371 mg, 0.70 mmol), palladium(II) acetate (78 mg, 0.35
mmol), and Et3N (483 μL, 3.48 mmol) were added, and the reaction
was stirred under reflux overnight. The solvent was removed to
dryness, and the residue was purified by flash chromatography on silica
gel (hexane/EtOAc 1:0 to 3:2) to obtain 24 (190 mg, 0.46 mmol,
1
27%) as a white solid: Rf 0.14 (hexane/EtOAc = 1:1); H NMR (500
MHz, DMSO-d6) δ 11.28 (s, 1H, Imid-NH), 10.73 (s, 1H, Amid-NH),
8.27 (d, J = 1.8 Hz, 1H, H-6), 8.12 (d, J = 1.3 Hz, 1H, H-4), 7.40−7.25
(m, 2H, Aryl-H), 7.05−6.90 (m, 3H, Aryl-H), 5.88 (dd, J = 10.6, 6.1
Hz, 1H, H-1′), 4.91 (t, J = 5.3 Hz, 1H, OH-5′), 4.78 (s, 2H, CH2),
4.15 (dd, J = 4.1, 2.6 Hz, 1H, H-4′), 3.83−3.64 (m, 2H, H-5′), 3.01
(dd, J = 17.8, 6.3 Hz, 1H, H-2′), 2.27 (dd, J = 17.9, 10.6 Hz, 1H, H-
2′); 13C NMR (126 MHz, DMSO-d6) δ 213.3 (C-3′), 168.9 (CO),
168.7 (CO), 167.6 (CO), 157.7, 144.0, 141.1, 134.5, 129.6 (Aryl-
CH), 122.6, 121.3 (Aryl-CH), 120.6 (C-4), 114.6 (Aryl-CH), 112.4
(C-6), 82.7 (C-4′), 71.8 (C-1′), 67.0 (CH2), 60.5 (C-5′), 44.3 (C-2′);
HRMS (ESI-TOF) m/z calcd for C21H17N2O7 [M − H]− 409.1041,
found 409.1029.
1-(5-(N-Phenoxyacetyl)amino-N-(dimethoxytrityl)-
p h t h a l i m i d - 3 - y l ) - 3 - O - [ ( 2 - c y a n o e t h o x y ) -
diisopropylaminophosphanyl]-5-O-(dimethoxytrityl)-1,2-di-
deoxy-β-D-ribofuranose (20). Under Ar atmosphere, 19 (187 mg,
0.18 mmol) was dissolved in anhyd CH2Cl2 (10.5 mL) and i-Pr2NEt
(110 μL, 0.60 mmol) was added. 2-Cyanoethoxy-N,N-di-iso-
propylaminophosphanyl chloride (103 μL, 0.46 mmol) was added,
and the reaction mixture was stirred at rt for 4 h. The mixture was
directly transferred on a silica gel column for purification (hexane/
EtOAc = 3:2, + 0.1% Et3N). Flash chromatography afforded 20 (150
mg, 0.12 mmol, 67%) as a white solid: Rf 0.52, 0.55 (hexane/EtOAc
1-(5-(N-Phenoxyacetyl)aminophthalimid-3-yl)-1,2-dideoxy-
β-D-ribofuranose (16). Method 1. Under Ar atmosphere, 15 (264
mg, 0.52 mmol) was dissolved in dry pyridine (5.3 mL). Phenoxyacetic
anhydride (224 mg, 0.78 mmol) was added, and the reaction mixture
was stirred at rt overnight. Water (5.3 mL) was added, and the mixture
was extracted first with hexane (2 × 25 mL, 1 × 30 mL) and then with
CH2Cl2 (1 × 20 mL). The organic layers were combined and
evaporated to dryness. The residue was taken up in anhyd THF (5.3
mL) under Ar atmosphere, and Et3N·3HF (679 μL, 4.17 mmol) was
added. The reaction mixture was stirred at rt overnight. A few drops of
ethanol were added, and THF was evaporated under reduced pressure.
The crude reaction mixture was purified by flash chromatography on
silica gel (CHCl3/EtOH = 5:1) to afford 16 (118 mg, 0.29 mmol,
55%) as a white solid. Method 2. In a degassed mixture of acetic acid
and acetonitrile (2:1, 2.6 mL), 24 (56 mg, 0.13 mmol) was dissolved
under Ar atmosphere at 0 °C. Sodium triacetoxyborohydride (43 mg,
0.20 mmol) was added, and the reaction mixture was stirred at 0 °C
for 1 h. A mixture of water and ethanol (1:1, 1.7 mL) was added and
the solvents were removed to dryness. The residue was purified by
flash chromatography on silica gel (CHCl3/EtOH 1:0 to 11:1)
yielding 16 (53 mg, 0.13 mmol, 94%) as a white solid: Rf 0.49
1
3:2); H NMR (600 MHz, DMSO-d6) δ 10.68−10.63 (2 × s, 1H,
Amid-NH), 8.13−8.05 (2 × d, J = 1.5 Hz, 1H, H-4), 7.99−7.94 (2 × d,
J = 1.8 Hz, 1H, H-6), 7.41−6.77 (m, 31H, Aryl-H), 5.61 (ddd, J =
16.2, 10.1, 5.6 Hz, 1H, H-1′), 4.72−4.70 (2 × s, 2H, CH2), 4.38−4.28
(m, 1H, H-3′), 4.08−3.99 (m, 1H, H-4′), 3.71−3.69 (4 × s, 12H,
OCH3), 3.67−3.58 (m, 2H, CH2), 3.55−3.46 [m, 2H, CH(CH3)2],
3.29−3.21 (m, 1H, H-5′), 3.16−3.11 (m, 1H, H-5′), 2.66 (t, J = 5.9
Hz, 1H, CH2), 2.52 (t, J = 1.8 Hz, 1H, CH2), 2.48−2.39 (m, 1H, H-
2′), 1.72−1.62 (m, 1H, H-2′), 1.12−1.08 [4 × s, 6H, CH(CH3)2)],
1.00−0.98 [4 × s, 6H, CH(CH3)2]; 31P NMR (243 MHz, DMSO-d6)
δ 147.46, 147.02; HRMS (ESI-TOF) m/z calcd for C72H74N4O12P [M
+ H]+ 1217.5035, found 1217.5126.
1β-(2,4-Dichloropyrimidin-5-yl)-1,2,3-trideoxy-3-oxo-D-ribo-
furanose (27). Dry CHCl3 (40 mL) was added to an argon-purged,
dried Schlenk flask containing 0.575 g (2.56 mmol, 0.10 equiv)
Pd(OAc)2 and 2.73 g (5.12 mmol, 0.20 equiv) P(PhF5)3. The mixture
was stirred for 1 h at room temperature before 5.90 g (25.6 mmol, 1.00
equiv) of 3-O-(tert-butyldimethylsilyl)-1,2-dideoxy-1,2-didehydro-D-
ribofuranose (10), 8.45 g (30.7 mmol, 1.20 equiv) of 2,4-dichloro-5-
iodopyrimidine (28), and 7.06 g (25.6 mmol, 1.00 equiv) of Ag2CO3
were added. The reaction mixture was stirred for 10 h at 70 °C. After
being cooled to rt, the reaction mixture was filtered on a pad of Celite
and eluted with CHCl3. Volatile compounds were removed under
reduced pressure, and the remaining brown oil was dissolved in 150
mL THF. At 0 °C, 0.5 mL of AcOH and 2 mL of a 1 M TBAF solution
in THF were added. After 1 h, the solvent was removed under reduced
pressure hexanes−EtOAc 2:1 to 1:1). Title compound 27 (2.26 g, 8.59
1
(CHCl3/EtOH = 5:1); H NMR (300 MHz, DMSO-d6) δ 11.22 (s,
1H, Imid-NH), 10.68 (s, 1H, Amid-NH), 8.17 (d, J = 1.7 Hz, 1H, H-
6), 7.99 (d, J = 1.7 Hz, 1H, H-4), 7.37−7.27 (m, 2H, Aryl-H), 7.04−
6.94 (m, 3H, Aryl-H), 5.74 (dd, J = 9.9, 5.7 Hz, 1H, H-1′), 5.15 (d, J =
3.7 Hz, 1H, OH-3′), 4.78−4.75 (m, 3H, OH-5′ + CH2), 4.22−4.15
(m, 1H, H-3′), 3.90−3.80 (m, 1H, H-4′), 3.59 (dt, J = 9.9, 4.7 Hz, 1H,
H-5′), 3.43 (dt, J = 11.2, 5.6 Hz, 1H, H-5′), 2.34 (dd, J = 12.3, 5.7 Hz,
1H, H-2′), 1.66 (ddd, J = 12.7, 10.0, 5.6 Hz, 1H, H-2′); 13C NMR
1
mmol, 34% yield) was obtained as a yellow oil: H NMR (500 MHz,
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CDCl3) δ 8.96 (s, 1H, H6), 5.46 (ddd, J = 10.5, 6.4, 0.8 Hz, 1H, H1′),
4.11 (t, J = 3.0 Hz, 1H, H4′), 4.02 (dd, J = 12.1, 2.9 Hz, 1H, H5′a),
4.00 (dd, J = 12.1, 3.2 Hz, 1H, H5′b), 3.16 (dd, J = 18.1, 6.4 Hz, 1H,
H2′a), 2.37 (dd, J = 18.1, 10.5 Hz, 1H, H2′b); 13C NMR (126 MHz,
CDCl3) δ 211.7 (C3′), 160.0 (C2/C4), 159.9 (C2/C4), 158.5 (C6),
132.0 (C5), 82.3 (C4′), 72.3 (C1′), 61.5 (C5′), 43.5 (C2′); HRMS
dried Schlenk flask was charged with 20 mL of dry 1,4-dioxane, 74 mg
(0.081 mmol, 0.04 equiv) of Pd2(dba)3, 141 mg (0.244 mmol, 0.12
equiv) of Xantphos, 492 mg (4.06 mmol, 2.00 equiv) of benzamide,
1.32 g (4.06 mmol, 2.00 equiv) of Cs2CO3, and 770 mg (2.03 mmol,
1.00 equiv) of 1β-(2,4-dichloropyrimidin-5-yl)-1,2-dideoxy-5-O-(tert-
butyldimethylsilyl)-D-ribofuranose 31. The mixture was stirred for 24 h
at 100 °C. After being cooled to rt, the reaction mixture was filtered on
a pad of Celite and eluted with MeOH. Volatiles were removed from
the filtrate under reduced pressure and the remaining crude product
was purified by flash chromatography on silica (gradient: hexanesa-
cetone 1:1 to 2:3). Title compound 32 (905 mg, 1.65 mmol, 81%) was
−
(ESI-FT-ICR) m/z calcd for C9H7Cl2N2O3 [M − H]+ 260.9839,
found 260.9853.
1β-(2,4-Dichloropyrimidin-5-yl)-1,2-dideoxy-D-ribofuranose
(28). A 2.26 g (8.59 mmol, 1.00 equiv) portion of 1β-(2,4-
dichloropyrimidin-5-yl)-1,2-dideoxy-D-ribofuranose 27 was dissolved
in 200 mL of CH3CN and 30 mL of AcOH. At 0 °C, 2.73 g (12.9
mmol, 1.50 equiv) of sodium triacetoxyborohydride was added slowly.
The reaction was monitored by TLC, and after 10 min, all starting
material was consumed. Thirty milliliters of 50 vol % aqueous EtOH
was added. Volatiles were removed under reduced pressure, and the
residue was purified by flash chromatography on silica (CH2Cl2/
MeOH 95:5). Nucleoside 28 (2.10 g, 7.92 mmol, 92% yield) was
isolated as a white foam: 1H NMR (500 MHz, CD3OD) δ 8.92 (d, J =
0.9 Hz, 1H, H6), 5.36 (dd, J = 10.0, 5.8 Hz, 1H, H1′), 4.37 (ddd, J =
6.5, 3.7, 1.6 Hz, 1H, H3′), 4.00 (td, J = 4.4, 2.6 Hz, 1H, H4′), 3.74 (dd,
J = 11.9, 4.1 Hz, 1H, H5′a), 3.71 (dd, J = 11.9, 4.8 Hz, 1H, H5′b), 2.50
(ddd, J = 13.0, 5.8, 2.1 Hz, 1H, H2′a), 1.90 (ddd, J = 13.0, 10.0, 5.9
Hz, 1H, H2′b); 13C NMR (126 MHz, CD3OD) δ 160.7 (C2/C4),
159.9 (C6), 159.9 (C2/C4), 135.1 (C5), 89.4 (C4′), 76.0 (C1′), 74.0
(C3′), 63.5 (C5′), 42.7 (C2′); HRMS (ESI-FT-ICR) m/z calcd for
1
isolated as a colorless solid: H NMR (500 MHz, CD3OD) δ 8.56 (s,
1H, H6), 8.13−8.05 (m, 2H), 8.05−7.98 (m, 2H), 7.72−7.62 (m, 2H),
7.63−7.51 (m, 4H), 5.41 (dd, J = 10.5, 5.2 Hz, 1H, H1′), 4.40 (dt, J =
5.8, 1.7 Hz, 1H, H3′), 4.13 (td, J = 3.8, 2.0 Hz, 1H, H4′), 3.76 (s, 1H,
H5′a), 3.76 (s, 1H, H5′b), 2.45 (ddd, J = 12.9, 5.3, 1.6 Hz, 1H, H2′a),
2.29−2.19 (m, 1H, H2′b), 0.82 (s, 9H, C(CH3)3), −0.00 (s, 3H, Si-
CH3), −0.08 (s, 3H, Si-CH3); 13C NMR (126 MHz, CD3OD) δ 167.4
(Camide), 167.1 (Camide), 158.1 (C2/C4), 158.0 (C2/C4), 157.8 (C6),
135.4 (Cq), 135.1 (Cq), 134.2, 133.7, 130.1, 129.8, 128.9, 128.9
(CHAr), 120.1 (C5), 89.8 (C4′), 77.4 (C1′), 73.8 (C3′), 64.8 (C5′),
42.2 (C2′), 26.3 (C(CH3)3), 18.2 (C(CH3)3), −5.3 (Si-CH3), −5.5
(Si-CH3);); HRMS (ESI-FT-ICR) m/z calcd for C29H35N4O5Si− [M
− H]+ 547.2382, found 547.2381.
1β-[2,4-Bis(benzoylamino)pyrimidin-5-yl]-1,2-dideoxy-D-ri-
bofuranose (33). 1β-[2,4-Bis(benzoylamino)pyrimidin-5-yl]-1,2-di-
deoxy-5-O-(tert-butyldimethylsilyl)-D-ribofuranose (32) (0.87 g, 1.55
mmol, 1.00 equiv) was dissolved in 25 mL of THF. NEt3·3HF (0.60
mL, 4.00 mmol, 2.60 equiv) was added dropwise. After 2 h, volatiles
were removed under reduced pressure, and the remaining solid was
purified by flash chromatography on silica (CH2Cl2/MeOH 9:1). 466
mg (1.07 mmol, 70% yield) of the title compound 33 were isolated as
+
C9H11Cl2N2O3 [M + H]+ 265.0141, found 265.0139.
1β-(2,4-Diaminopyrimidin-5-yl)-1,2-dideoxy-D-ribofuranose
(2). At −78 °C, ammonia was condensed into 40 mL of dry methanol.
1β-(2,4-dichloropyrimidin-5-yl)-1,2-dideoxy-D-ribofuranose (28)
(0.670 g, 2.53 mmol) was added, and the solution was transferred
to a glass reactor. The sealed reactor was heated to 120 °C for 12 h.
After the mixture was cooled to rt, volatiles were removed under
reduced pressure, and the crude product was purified by flash
chromatography on silica (gradient: CH2Cl2/MeOH 9:1 to 65:35, +
5% H2O). Title compound 2 (0.414 g, 1.83 mmol, 72% yield) was
1
white solid: H NMR (300 MHz, DMSO-d6) δ 11.06 (s, 1H, NH),
10.87 (s, 1H, NH), 8.91 (s, 1H), 8.15−7.86 (m, 4H), 7.72−7.35 (m,
6H), 5.12 (dd, J = 10.1, 5.6 Hz, 1H), 5.05 (d, J = 3.9 Hz, 1H), 4.85 (t,
J = 5.6 Hz, 1H), 4.23−4.14 (m, 1H), 3.75 (td, J = 4.7, 2.3 Hz, 1H),
3.49 (m, 2H, H5′a + H5′b), 2.21 (ddd, J = 12.8, 5.7, 1.7 Hz, 1H,
H2′a), 1.89 (ddd, J = 12.7, 10.1, 5.8 Hz, 1H, H2′b); 13C NMR (75
MHz, DMSO-d6) δ 166.1 (Camide), 165.6 (Camide), 158.3 (C6), 157.1
(C2/C4), 156.4 (C2/C4), 134.1 (Cq), 133.0 (Cq), 132.5, 132.1
(CHAr), 128.6, 128.4, 128.2, 128.1 (CHAr), 126.2 (Cq), 109.6 (C5),
87.6 (C4′), 74.0 (C1′), 72.1 (C3′), 62.1 (C5′), 41.6 (C2′); HRMS
1
isolated as a yellow foam: H NMR (300 MHz, CD3OD) δ 7.64 (s,
1H, H6), 5.00−4.72 (m, 1H, H1′), 4.34 (dt, J = 6.2, 1.7 Hz, 1H, H3′),
3.88 (q, J = 2.6 Hz, 1H, H4′), 3.75−3.64 (m, 1H, H5′a), 3.70−3.58
(m, 1H, H5′b), 2.13 (ddd, J = 13.1, 11.0, 6.0 Hz, 1H, H2′a), 1.95
(ddd, J = 13.2, 5.5, 1.4 Hz, 1H, H2′b); 13C NMR (75 MHz, CD3OD)
δ 164.7 (C2/C4), 156.9 (C2/C4), 141.7 (C6), 109.5 (C5), 89.5
(C4′), 77.8 (C1′), 74.3 (C3′), 62.9 (C5′), 40.9 (C2′); HRMS (ESI-
+
(ESI-FT-ICR) m/z calcd for C23H22N4NaO5 [M + Na]+ 457.1482,
+
FT-ICR) m/z calcd for C9H15N4O3 [M + H]+ 227.1139, found
found 457.1485.
227.1137.
1β-[2,4-Bis(benzoylamino)pyrimidin-5-yl]-1,2-dideoxy-5-O-
(4,4′-dimethoxytriphenylmethyl)-D-ribofuranose (34). 1β-[2,4-
Bis(benzoylamino)pyrimidin-5-yl]-1,2-dideoxy-D-ribofuranose (33)
(434 mg, 1.00 mmol, 1.00 equiv) was dissolved in 12 mL of dry
pyridine in an argon-purged, dried Schlenk flask. 4,4′-Dimethoxytrityl
chloride (510 mg, 1.50 mmol, 1.50 equiv) was added slowly, and the
resulting solution was stirred for 2 d at rt The reaction was quenched
with 5 mL of MeOH, and volatile compounds were removed under
reduced pressure. The remaining crude product was purified by flash
chromatography on silica (hexanes/acetone 2:3, + 0.1% ethyldimethyl-
amine) yielding 690 mg (0.93 mmol, 93% yield) of the title compound
34 as yellow foam: 1H NMR (300 MHz, CDCl3) δ 10.55 (s, 1H, NH),
9.91 (s, 1H, NH), 8.42 (s, 1H, H6), 8.04−7.91 (m, 2H), 7.89−7.72
(m, 2H), 7.63−7.27 (m, 8H), 7.24−7.08 (m, 7H), 6.85−6.58 (m, 4H),
5.27 (dd, J = 10.4, 4.9 Hz, 1H, H1′), 4.58 (br s, 1H, OH), 4.42 (br s,
1H, H3′), 4.33 (t, J = 3.1 Hz, 1H, H4′), 3.69 (s, 3H, OCH3), 3.69 (s,
3H, OCH3), 3.25 (dd, J = 10.2, 5.2 Hz, 1H, H5′a), 3.21 (dd, J = 10.2,
4.5 Hz, 1H, H5′b), 2.53 (dd, J = 12.9, 5.1 Hz, 1H, H2′a), 2.37−2.23
(m, 1H, H2′b); 13C NMR (75 MHz, CDCl3) δ 165.4 (Camide), 165.0
(Camide), 158.6 (CAr-O), 157.5 (C2/C4), 157.2 (C2/C4), 156.4 (C6),
144.5, 135.7, 133.8 (Cq), 133.0, 132.4, 130.0, 129.1, 128.7, 127.6, 113.2
(CHAr), 110.1 (C5), 87.5 (C4′), 86.4 (Cq), 76.1 (C1′), 73.5 (C3′),
64.4 (C5′), 55.3 (OCH3), 40.0 (C2′); HRMS (ESI-FT-ICR) m/z
1β-(2,4-Dichloropyrimidin-5-yl)-1,2-dideoxy-5-O-(tert-butyl-
dimethylsilyl)-D-ribofuranose (31). An argon-purged, dried
Schlenk flask was charged with 20 mL of dry DMF, 404 mg (5.94
mmol, 2.50 equiv) of imidazole, and 630 mg (2.38 mmol, 1.00 equiv)
of 1β-(2,4-dichloropyrimidin-5-yl)-1,2-dideoxy-D-ribofuranose (28).
To that solution was slowly added 430 mg (2.85 mmol, 1.20 equiv)
of TBDMSCl. The reaction mixture was stirred at rt for 4 h, and then
50 mL water was added. The aqueous phase was washed three times
with 100 mL of ethyl acetate and dried over MgSO4, and volatile
compounds were removed under reduced pressure. The remaining
crude product was purified by flash chromatography on silica
(hexanes/EtOAc 3:1). Title compound 31 (770 mg, 2.00 mmol,
85% yield) was isolated as a colorless oil: 1H NMR (300 MHz,
CDCl3) δ 8.82 (s, 1H, H6), 5.37 (dd, J = 9.9, 6.0 Hz, 1H, H1′), 4.49
(ddt, J = 5.7, 2.2, 1.1 Hz, 1H, H3′), 4.07 (ddd, J = 4.4, 3.2, 2.3 Hz, 1H,
H4′), 3.83 (dd, J = 10.8, 3.3 Hz, 1H, H5′a), 3.77 (dd, J = 10.8, 4.4 Hz,
1H, H5′b), 2.52 (ddd, J = 13.0, 5.9, 2.0 Hz, 1H, H2′a), 1.88 (ddd, J =
13.0, 9.9, 5.7 Hz, 1H, H2′b), 0.86 (s, 9H, C(CH3)3), 0.07 (s, 6H, Si-
CH3); 13C NMR (75 MHz, CDCl3) δ 159.6 (C2/C4), 159.3 (C2/
C4), 158.6 (C6), 133.6 (C5), 87.8 (C4′), 75.0 (C1′), 74.4 (C3′), 63.8
(C5′), 42.2 (C2′), 26.0 (C(CH3)3), 18.4 (C(CH3)3), −5.3 (Si-CH3),
−5.4 (Si-CH3); HRMS (ESI-FT-ICR) m/z calcd for
C15H25Cl2N2O3Si+ [M + H]+ 379.1006, found 379.1007.
+
calcd for C44H40N4NaO7 [M + Na]+ 759.2789, found 759.2790.
1β-[2,4-Bis(benzoylamino)pyrimidin-5-yl]-1,2-dideoxy-5-O-
(tert-butyldimethylsilyl)-D-ribofuranose (32). An argon-purged,
1β-[2,4-Bis(benzoylamino)pyrimidin-5-yl]-1,2-dideoxy-5-O-
(4,4′-dimethoxytriphenylmethyl)-D-ribofuranose-3-[(2-
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cyanoethyl)(N,N-diisopropyl)]phosphoramidite (35). 1β-[2,4-
Bis(benzoylamino)pyrimidin-5-yl]-1,2-dideoxy-5-O-(4,4′-dimethoxy-
triphenylmethyl)-D-ribofuranose (34) (170 mg, 0.23 mmol, 1.00
equiv) and 0.12 mL (0.69 mmol, 3.00 equiv) of diisopropylethylamine
were dissolved in 5 mL of dry CH2Cl2 in an argon-purged, dried
Schlenk flask. 2-Cyanoethyl N,N-diisopropylchlorophosphoramidite
(0.08 mL, 0.35 mmol, 1.50 equiv) was added and the solution stirred
for 2 h. Volatiles were removed under reduced pressure, and the crude
product was purified by flash chromatography on silica (hexanes/
acetone 2:1, + 0.1% ethyldimethylamine). Title compound 35 (130
mg, 0.14 mmol, 60% yield) was isolated as a colorless solid: 31P NMR
(121 MHz, DMSO-d6) δ 147.7, 147.2; HRMS (ESI-FT-ICR) m/z
calcd for C53H58N6O8P+ [M + H]+ 937.4048, found 937.4048.
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Oligonucleotides (DNA1, DNA2). DNA1 was synthesized on a
DNA synthesizer with standard phosphoramidite protocols for the
ultramild synthesis method using CPGs (1 μmol) with longer coupling
times of 625 s and 0.1 M concentration of phosphoramidite 20. A
special capping mix (THF/pyridine/Pac2O) for ultramild synthesis
was applied. The chemicals for the DNA synthesis were purchased.
After preparation, the trityl-off oligonucleotide was cleaved off the
resin and deprotected with 1 mL of 0.05 M K2CO3 in MeOH at 65 °C
for 48 h. After cooling, 6 μL of acetic acid was added, and the
oligonucleotide was dried and purified by reversed-phase HPLC using
the following conditions: A = NH4OAc buffer (50 mM), pH = 6.5; B =
MeCN; gradient = 0−12% B over 45 min. Oligonucleotide DNA2 was
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35. Deprotection was achieved under standard conditions using concd
ammonia at elevated temperature. Unmodified oligonucleotides were
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their absorbance at 260 nm on a spectrophotometer. Duplexes were
prepared by heating the modified oligonucleotide in presence of 1.05
equiv. of unmodified counter strand to 90 °C, followed by cooling to
room temperature. All melting temperatures were determined by the
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change of absorbance at 260 nm (1: ε260 = 9,300 M−1 cm−1, 2: ε260
=
2,200 M−1 cm−1) using the following conditions: 2.5 μM DNA, 10
mM Na−Pi buffer, pH = 7.0, 250 mM NaCl; heating/cooling rate 0.7
°C/min, data interval 0.5 °C.
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* Supporting Information
Images of NMR spectra and MS analysis of compounds; HPLC
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Corresponding Author
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Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
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