Synthesis of Homo-C-Nucleoside Phosphoramidites and Their Site-Specific Incorporation into Oligonucleotides

Article abstract of DOI:10.1002/ejoc.201500996

An efficient procedure was developed to prepare homo-C-nucleosides. The β-allyl C-glycoside of D-ribose was transformed into the thienopyrimidine nucleoside and the benzodiazepine nucleoside of 2-deoxy-D-ribose. For incorporation into oligonucleotides by solid-phase synthesis, both derivatives were transformed into the related 3′-phosphoramidite building blocks. These phosphoramidites were then site-specifically incorporated into DNA oligonucleotides. The modified DNA strands were hybridized with different DNA and RNA strands, and their melting temperatures and circular dichroism spectra were studied. Interestingly, although in the case of DNA-DNA hybrids the nonnatural nucleosides caused a marked decrease in melting temperature, to levels even lower than those for mismatched hybrids, in the case of DNA-RNA hybrids very similar melting temperatures were measured for the nonnatural nucleosides and the unmodified oligonucleotides. 3′-Phosphoramidites of thienopyrimidine and benzodiazepine homo-C-nucleosides were prepared and site-specifically incorporated into DNA. These modified oligonucleotides were hybridized with different DNA and RNA strands in order to study their duplex characteristics in terms of their melting temperatures and circular dichroism spectra.

Full text of DOI:10.1002/ejoc.201500996

FULL PAPER
DOI: 10.1002/ejoc.201500996
Synthesis of Homo-C-Nucleoside Phosphoramidites and Their Site-Specific
Incorporation into Oligonucleotides
Katharina Höfler,[a] Tristan Zimmermann,[a] Dilver Peña Fuentes,[b,c] Christian Vogel,*[c] and
Chris Meier*[a]
Keywords: Nucleobases / Nucleosides / Oligonucleotides / Nitrogen heterocycles / DNA
An efficient procedure was developed to prepare homo-C-
nucleosides. The β-allyl C-glycoside of -ribose was trans-
and RNA strands, and their melting temperatures and circu-
lar dichroism spectra were studied. Interestingly, although in
D
formed into the thienopyrimidine nucleoside and the benzo- the case of DNA–DNA hybrids the nonnatural nucleosides
diazepine nucleoside of 2-deoxy-
D
-ribose. For incorporation
caused a marked decrease in melting temperature, to levels
even lower than those for mismatched hybrids, in the case of
DNA–RNA hybrids very similar melting temperatures were
measured for the nonnatural nucleosides and the unmodified
oligonucleotides.
into oligonucleotides by solid-phase synthesis, both deriva-
tives were transformed into the related 3Ј-phosphoramidite
building blocks. These phosphoramidites were then site-
specifically incorporated into DNA oligonucleotides. The
modified DNA strands were hybridized with different DNA
side analogue. In contrast, the natural N,O-acetal can be
cleaved enzymatically. Beyond the N- and C-nucleoside
structures, a further variant involves the introduction of a
carbon atom or a short linker between the heterocyclic base
and the glycon moiety. This leads to the formation of
homo-C-nucleosides.[1,2]
Introduction
Research on nucleoside analogues has been going on for
many years, and there is still a high interest in new nucleo-
side analogues, e.g., as artificial nucleosides, or as potential
antiviral, antibacterial, or anticancer compounds. For ex-
ample, in antiretroviral therapy, nucleoside analogues are
clinically used as strong inhibitors of viral polymerases in
the treatment of HIV and HCV (hepatitis C). To express
antiviral activity, nucleoside analogues have to be trans-
formed intracellularly by kinases into their 5Ј-triphosphate
forms, which then interact with the polymerase. In recent
decades, a large number of nucleoside analogues have been
described in which the sugar moiety and/or in heterocyclic
ring have been modified. In addition to nucleoside ana-
logues in which the heterocyclic base is linked to the glycon
part by an N,O-acetal, C-nucleosides are known in which
the anomeric centre is covalently linked to a carbon atom
of the base analogue. In these compounds, there is a stable
linkage between the two fragments that make up the nucleo-
The design and synthesis of homo-C-nucleosides has
been carried out for a number of different reasons. Some of
the aims behind work on these compounds are as follows:
the synthesis of homo-C-nucleosides with potential bio-
logical activity;[3–5] the synthesis of tools for elucidating the
structural and functional properties of damaged DNA;[6] to
illustrate the hybridizations and conformational changes of
DNA and RNAs;[7] and to give an answer to the question
as to why DNA evolved on earth to have the structure that
it does.[8–12] Since changes in the structure of nucleic acids
control a large number of biological processes, artificial nu-
cleic acids might be a target of therapeutic application.[13–15]
In this context, controlling the binding specificity, thermal
and enzymatic stability of artificial oligonucleotides is still
a challenge. Because of the higher conformational flexibility
of the bonds between the glycon residue and the heterocy-
clic ring in homo-C-nucleosides, we became interested in
the potentially modified properties after the incorporation
of such compounds into oligonucleotides. Thus, in this pa-
per we describe the synthesis of homo-C-nucleosides, their
conversion into the corresponding 3Ј-phosphoramidites,
and the properties of the homo-C-nucleoside-containing
oligonucleotides.
[a] Organic Chemistry, Department of Chemistry, Faculty of
Sciences, University of Hamburg,
Martin-Luther-King-Platz 6, 20146 Hamburg, Germany
E-mail: chris.meier@chemie.uni-hamburg.de
http://www.chemie.uni-hamburg.de/oc/meier/index.html
[b] Universidad de Granma,
Carretera Manzanillo Km. 17.5, Bayamo Granma, Cuba
[c] University of Rostock, Institute of Chemistry,
Albert-Einstein-Str. 3a, 18059 Rostock, Germany
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201500996.
Eur. J. Org. Chem. 2015, 6841–6849
© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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K. Höfler, T. Zimmermann, D. Peña Fuentes, C. Vogel, C. Meier
FULL PAPER
Results and Discussion
Chemistry
presence of imidazole in DMF at low temperature to
achieve regioselective silylation. This resulted in compound
2 (Scheme 1). Iodination of alcohol 2, followed by radical-
mediated reduction with Bu3SnH gave TIPS-protected allyl-
2Ј-deoxyriboside 3 in 70% overall yield from 1.[16] Oxidative
hydroboration of alkene 3 led first to alcohol 4, which was
then selectively oxidized to give aldehyde 5 (84% overall
yield).[17] Treatment of 5 with malononitrile gave the corre-
sponding Knoevenagel product (i.e., 6) in 65% yield. On the
other hand, reaction of 5 with phenylethynyllithium gave a
pentynol with a new stereogenic centre. However, this ste-
reogenic centre was lost after oxidation to give ethynyl
ketone 7 in 62% overall yield.[17]
Synthesis of a Knoevenagel Product and an Acetylenic
Ketone as Versatile Intermediates for the Synthesis of
Nucleoside Analogues
Pursuing a programme directed towards the synthesis of
homo-C-nucleosides, we have previously described an ef-
ficient route for the preparation of β-allyl C-glycosides of
d-ribofuranose and 2-deoxy-d-ribofuranose.[16] To prevent
the formation of the pyranose form, the furanose form was
fixed by condensation of d-ribose with acetone to give 2,3-
O-isopropylidene-d-ribofuranose. After acetylation, the di-
acetate was converted into C-allyl glycoside 1 by treatment
with allyltrimethylsilane (AllTMS) and zinc bromide in
CH3NO2 as solvent. Pure protected β-C-allyl glycoside 1
was isolated in 85% yield.[16] After complete deprotection
of compound 1, the resulting allylriboside was treated with
1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (TIPSCl) in
Synthesis of the 3Ј-Phosphoramidite of Thienopyrimidine-
2Ј-deoxyriboside Nucleoside
Treatment of 6 with elemental sulfur and trimethylamine
in N,N-dimethylformamide (DMF) gave aminothiophene 8
as a pale yellow solid in 93% yield. For the synthesis of
4-aminothienopyrimidine 9, compound 8 was treated with
triethyl orthoformate under reflux for 2 h. Without purifi-
cation, the resulting formimidate was treated with saturated
ethanolic ammonia solution to give compound 9 in 89%
overall yield.
After benzoylation of the amino group (10, 83%), the
TIPS protecting group was removed by treatment with
tetra-n-butylammonium fluoride (TBAF) to give diol 11 in
95% yield. Next, regioselective protection of the primary
5Ј-hydroxyl group was achieved by treatment of 11 with a
slight excess of 4,4Ј-dimethoxytrityl chloride (DMTrCl) in
dry DMF in the presence of dimethylaminopyridine
(DMAP) and triethylamine. After 24 h, the process was re-
Scheme 1. Synthesis of Knoevenagel product 6 and acetylenic
ketone 7 starting from d-ribose β-allyl C-glycoside 1. Reagents and
Scheme 2. Synthesis of 3Ј-phosphoramidite 13 from Knoevenagel
product 6. Reagents and conditions: (i) sulfur, NEt3, DMF, 2 h,
conditions: (i) HCl (aq.), EtOH, 2 d, 20 °C; (ii) TIPSCl, imidazole, room temp.; (ii) HC(OEt)3, 2 h, reflux, then ethanol/ammonia, 2 h,
DMF, 1 h, 5 °C; (iii) I2, Ph3P, imidazole, dry toluene, then Bu3SnH,
AIBN, dry toluene 10 h, reflux; (iv) B2H6·THF, 3 h, 0 °C, followed
by H2O2; (v) Dess–Martin oxidation; (vi) malononitrile, Al2O3, tol-
uene, 3 d, reflux; (vii) phenylethynyllithium, dry THF, 4 h, 20 °C.
reflux; (iii) BzCl, pyridine, 12 h, 0 °C – 20 °C; (iv) TBAF, acetone,
2 h, room temp.; (v) DMTrCl, DMAP, NEt3 (in two portions),
DMF, 2ϫ 24 h, room temp.; (vi) 2-cyanoethyl-N,N-diisoprop-
ylchlorophosphoramidite, DIPEA, CH2Cl2, 6 h, room temp.
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Synthesis of Homo-C-Nucleoside Phosphoramidites
peated, and compound 12 was isolated in 83% yield. Fi- d(GTA GAA TTC TAC)-3Ј] 19a (the modification site is
nally, compound 12 was converted into 3Ј-phosphoramidite highlighted in bold). Cleavage of the 12-mer oligonucleo-
13 in 80% yield by treatment with 2-cyanoethyl-N,N-di- tides from the solid support and removal of the protecting
isopropylchlorophosphoramidite and diisopropylethyl- groups was achieved by treatment with ammonia for 2 h at
amine (DIPEA) in dry CH2Cl2 as solvent (Scheme 2).
45 °C.
In addition, the complementary RNA strand [5Ј-(GAU
Synthesis of the 3Ј-Phosphoramidite of Benzodiazepine-2Ј-
deoxyriboside Nucleoside
GGC GCC GAG)-3Ј] 20 to the NarI sequence was synthe-
sized for further thermal stability assays. The DNA oligo-
nucleotide products were purified by reverse-phase HPLC,
while the RNA oligonucleotides were purified by anion-ex-
change HPLC. The prepared oligonucleotides were charac-
terized by MALDI-TOF mass spectrometry (negative
mode).
Cyclization to diazepine 14 was achieved in 71% yield
when a mixture of ethynyl ketone 7 and benzene-1,2-di-
amine was heated under reflux in ethanol (EtOH) for 5 h.
TIPS-deprotection of compound 14 gave diol 15 (94%),
and subsequent introduction of a 5Ј-DMTr group led to
compound 16 in 65% yield. The desired 3Ј-phosphoramid-
ite (i.e., 17) was obtained in 76% yield by treatment of
alcohol 16 with 2-cyanoethyl-N,N-diisopropylchlorophos-
phoramidite (Scheme 3). The conditions for the last three
reaction steps were identical to those described for the syn-
thesis of amidite 13.
Properties of the Modified Oligonucleotides
Melting Temperature (Tm) and Circular Dichroism Studies
To compare the effect on the thermal stability of oligonu-
cleotide hybrids containing a thienopyrimidine nucleoside
or a benzodiazepine nucleoside, first mismatched NarI oli-
gonucleotide duplexes were formed. As a reference NarI du-
plex, annealing of the NarI-sequence [5Ј-d(CTC GGC GCC
ATC)-3Ј] 18 and complementary [5Ј-d(GAT GGC GCC
GAG)-3Ј] 21a was measured (Tm = 60.5 °C; Table 1). Next,
oligonucleotide 18 was hybridized with strands 21b–21d,
each containing a natural nucleoside other than the canoni-
cal one. Moreover, an oligonucleotide 21e containing an
ABASIC site in place of the canonical base was also in-
cluded. The corresponding UV melting-temperature data
for the mismatched NarI oligonucleotides is given in
Table 1.
Table 1. Tm values of NarI-oligonucleotide 5Ј-d(CTC GGC GCC
ATC)-3Ј 18 with 21a–21e.[a]
Oligonucleotide
Tm [°C]
5Ј-d(GAT GGC GCC GAG)-3Ј 21a
5Ј-d(GAT GGG GCC GAG)-3Ј 21b
5Ј-d(GAT GGA GCC GAG)-3Ј 21c
5Ј-d(GAT GGT GCC GAG)-3Ј 21d
5Ј-d(GAT GG[ABASIC] GCC GAG)-3Ј 21e
60.5
50.9
48.2
50.2
39.5
Scheme 3. Synthesis of 3Ј-phosphoramidite 17 from ethynyl ketone
7. Reagents and conditions: (i) benzene-1,2-diamine, EtOH, 5 h, re-
flux; (ii) TBAF, acetone, 2 h, room temp.; (iii) DMTrCl, DMAP,
NEt3 (in two portions), DMF, 2ϫ 24 h, room temp.; (iv) 2-cyano-
ethyl-N,N-diisopropylchlorophosphoramidite, diisopropylamine,
CH2Cl2, 6 h, room temp.
[a] Conditions: unmodified 5Ј-d(CTC GGC GCC ATC)-3Ј oligonu-
cleotide 18 (1 nmol), complementary mismatched oligonucleotide
(1 nmol), phosphate buffer (pH 6.8; 10 mm), NaCl (140 mm), ethyl-
enediaminetetraacetic acid (EDTA; 1.0 mm).
Incorporation of Phosphoramidites into DNA
Oligonucleotides
For mismatched oligonucleotides 21b–21d, a decrease of
3Ј-Phosphoramidites 13 and 17 were dissolved in aceto- 9.5–12.3 °C was observed relative to the Tm value of 60.5 °C
nitrile (0.1 m solutions), and were site-specifically incorpo- for the NarI reference (i.e., 21a). In fact, for the duplex
rated into DNA oligonucleotides using standard automated formed by 18 and ABASIC-site-containing oligonucleotide
solid-phase synthesis. Commercially available 3Ј-phos- 21e, a thermal destabilization of 21.0 °C was determined.
phoramidites were used for the normal nucleosides, as well
To study the base-pairing properties of homo-C-nucleo-
as for the ABASIC nucleoside analogue. Quantitative cou- side-containing oligonucleotides, we recorded the melting
pling efficiency was obtained when the modified 3Ј-phos- temperatures of oligonucleotides in which a thienopyrimid-
phoramidites were added in two coupling steps and with an ine or benzodiazepine nucleoside is hybridized with each
extended coupling time of 199 s.
of the four natural nucleosides. All of these melting curves
Two different site-specifically modified DNA sequences showed a hyperchromic effect with single melting transi-
were prepared: NarI oligonucleotide [5Ј-d(CTC GGC GCC tions. The data for NarI sequence 22, containing a thieno-
ATC)-3Ј] 18, and palindromic EcoRI oligonucleotide [5Ј- pyrimidine nucleoside, are listed in Table 2, and the results
Eur. J. Org. Chem. 2015, 6841–6849
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K. Höfler, T. Zimmermann, D. Peña Fuentes, C. Vogel, C. Meier
FULL PAPER
for NarI sequence 23, containing a benzodiazepine nucleo- nucleosides and any of the natural nucleobases. Interest-
side, are given in Table 3.
ingly, both of the duplexes containing the “nonnatural”
bases and with an ABASIC site opposite the modification
showed higher Tm values (Table 2; Tm = 43.6 °C; Table 3,
Tm = 47.5 °C; compound 21e) than the NarI-duplex modi-
fied only by the presence of an ABASIC site (Table 1; Tm
= 39.5 °C; compound 21e). This indicates that the introduc-
tion of the ABASIC site compensates for the distortion in-
troduced into the double helix by the nonnatural base.
In addition, the thienopyrimidine- and benzodiazepine-
nucleoside-containing oligonucleotides (i.e., 22 and 23,
respectively) were hybridized with a complementary RNA
20 to give RNA–DNA hybrids. Tm value data are summa-
Table 2. Tm values of thienopyrimidine-nucleoside-containing
NarI-oligonucleotide 5Ј-d(CTC GGC [Thieno]CC ATC)-3Ј 22 with
21a–21e.[a]
Oligonucleotide
Tm [°C]
5Ј-d(GAT GGC GCC GAG)-3Ј 21a
5Ј-d(GAT GGG GCC GAG)-3Ј 21b
5Ј-d(GAT GGA GCC GAG)-3Ј 21c
5Ј-d(GAT GGT GCC GAG)-3Ј 21d
5Ј-d(GAT GG[ABASIC] GCC GAG)-3Ј 21e
46.3
43.3
47.5
49.6
43.6
[a] Conditions: 5Ј-d(CTC GGC [Thieno]CC ATC)-3Ј oligonucleo-
tide 22 (1 nmol), complementary oligonucleotide (1 nmol), phos-
phate buffer (pH 6.8; 10 mm), NaCl (140 mm), ethylenediamine- rized in Table 4. As references, an RNA–RNA hybrid (Tm
tetraacetic acid (EDTA; 1.0 mm).
= 71.4 °C) and an RNA–DNA hybrid (Tm = 63.7 °C) were
also studied. A decrease in thermal stability for the thieno-
pyrimidine- or the benzodiazepine-nucleoside-containing
Table 3. Tm values of benzodiazepine-nucleoside-containing NarI-
oligonucleotide 5Ј-d(CTC GGC [Benzo]CC ATC)-3Ј 23 with 21a– DNA–RNA hybrids compared to the RNA–RNA reference
21e.[a]
(Tm = 71.4 °C) of 7.2–10.1 °C was observed. Interestingly,
the differences in Tm values between the thienopyrimidine-
or benzodiazepine-containing DNA–RNA duplexes and
the unmodified DNA–RNA reference duplex (20 with 18;
Tm = 63.7 °C) were very small. Here, the ABASIC site
showed the lowest Tm value (Tm = 60.9 °C).
Oligonucleotide
Tm [°C]
5Ј-d(GAT GGC GCC GAG)-3Ј 21a
5Ј-d(GAT GGG GCC GAG)-3Ј 21b
5Ј-d(GAT GGA GCC GAG)-3Ј 21c
5Ј-d(GAT GGT GCC GAG)-3Ј 21d
5Ј-d(GAT GG[ABASIC] GCC GAG)-3Ј 21e
42.6
42.1
43.2
46.9
47.5
Table 4. Tm values of RNA NarI-oligonucleotide 5Ј-(GAU GGC
GCC GAG)-3Ј 20.[a]
[a] Conditions: 5Ј-d(CTC GGC [Benzo]CC ATC)-3Ј oligonucleo-
tide 23 (1 nmol), complementary oligonucleotide (1 nmol), phos-
phate buffer (pH 6.8; 10 mm), NaCl (140 mm), ethylenediamine-
tetraacetic acid (EDTA; 1.0 mm).
Oligonucleotide
Tm [°C]
5Ј-(CUC GGC GCC AUC)-3Ј 24
71.4
63.7
61.3
64.2
60.9
5Ј-d(CTC GGC GCC ATC)-3Ј 18
Compared to the Tm value of the NarI reference duplex
formed by strand 18 and 21a (Tm = 60.5 °C), the Tm values
for the thienopyrimidine-nucleoside-containing NarI se-
quences (Table 2) were found to be lower by 10.9–17.2 °C.
However, the lowest Tm value was determined when dG was
placed opposite to the thienopyrimidine nucleoside modifi-
cation. The Tm value of the duplex containing a T opposite
the thienopyrimidine nucleoside was 6.3 °C higher than that
of the dG-thienopyrimidine nucleoside modification men-
tioned above, and was very similar to the Tm of a “normal”
mismatched base pairing (Table 1).
Despite the different structures of the homo-C-nucleo-
sides, the oligonucleotides containing a benzodiazepine nu-
cleoside (Table 3) showed destabilization of the Tm values
by 13.6–18.4 °C, which is similar to the results obtained for
the thienopyrimidine-nucleoside-containing oligonucleo-
tides. Again, the positioning of thymidine opposite the non-
natural base caused the least destabilisation, and this du-
plex was almost as stable as the duplex with an ABASIC
site opposite the “nonnatural” base. Compared to the re-
sults with the mismatched NarI duplexes (Table 1), the ther-
mal destabilization of the modified duplexes was even more
significant, which points to a severe local distortion of the
DNA double helix.
5Ј-d(CTC GGC [Thieno]CC ATC)-3Ј 22
5Ј-d(CTC GGC [Benzo]CC ATC)-3Ј 23
5Ј-d(CTC GGC [ABASIC]CC ATC)-3Ј 25
[a] Conditions: RNA NarI-oligonucleotide 5Ј-(GAU GGC GCC
GAG)-3Ј oligonucleotide 20 (1 nmol), complementary oligonucleo-
tide (1 nmol), phosphate buffer (pH 6.8; 10 mm), NaCl (140 mm),
ethylenediaminetetraacetic acid (EDTA; 1.0 mm).
For the palindromic EcoRI-oligonucleotides, compounds
13 and 17 were introduced at two different positions. The
first position, at the 5Ј-terminus, is situated inside the re-
cognition sequence of the restriction enzyme EcoRI,
whereas position 4 (from the 5Ј terminus) is outside the en-
zymatic cleavage site. The melting temperatures are summa-
rized in Table 5.
Table 5. Tm values of self-complementary EcoRI-oligonucleotides
19a–19e.[a]
Oligonucleotide
Tm [°C]
5Ј-d(GTA GAA TTC TAC)-3Ј 19a
35.1
18.2
16.4
20.4
17.4
5Ј-d([Thieno]TA GAA TTC TAC)-3Ј 19b
5Ј-d(GTA [Thieno]AA TTC TAC)-3Ј 19c
5Ј-d([Benzo]TA GAA TTC TAC)-3Ј 19d
5Ј-d(GTA [Benzo]AA TTC TAC)-3Ј 19e
[a] Conditions: EcoRI-oligonucleotide 19a–19e (2 nmol), phos-
phate buffer (pH 6.8; 10 mm), NaCl (140 mm), ethylenediamine-
tetraacetic acid (EDTA; 1.0 mm).
The differences in the Tm values that result from varying
the natural nucleobases opposite the thienopyrimidine and
benzodiazepine moieties within the modified oligonucleo-
Although one should keep in mind that in this experi-
tide duplexes are small. This indicates that there are no ment two modifications were present in the duplex because
strong stacking interactions between either of the homo-C- of the self-complementarity of the singly modified oligonu-
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Synthesis of Homo-C-Nucleoside Phosphoramidites
cleotides, a significant decrease in the Tm values of the thi-
The maxima in the CD spectra of oligonucleotides 19c
enopyrimidine- or benzodiazepine-containing EcoRI-se- and 19e, containing modifications at position 4, were found
quences (i.e., 19b–19e) of 14.7–18.7 °C compared to the ref- to be shifted to shorter wavelengths. In addition, for the
erence duplex (i.e., 19a) with a Tm of 35.1 °C was deter- NarI oligonucleotides, no overall conformational difference
mined. The difference between thienopyrimidine- or benzo- was observed. For oligonucleotides containing an adenine
diazepine-containing duplexes with modifications at posi- C-nucleoside, Ishiyama et al. also observed a right-handed
tion 1 and duplexes with modifications at position 4 is conformation, whereas for homonuclear C-nucleoside oli-
small, although in both cases the internal modification re- gonucleotides, an unusual left-handed helix type was ob-
sulted in a lower Tm value. This is to be expected, as a result served.[2]
of the more pronounced distortion when the modification is
situated internally. In none of the cases could any enzymatic
Conclusions
restriction of the EcoRI sequences be observed. In contrast,
We have described an experimental procedure for the
using the same experimental conditions, we have previously
synthesis of 3Ј-phosphoramidites of homo-C-nucleoside an-
shown that the unmodified EcoRI duplex sequence was
alogues.
readily cleaved to give the expected fragments.[18] It remains
These phosphoramidites were site-specifically incorpo-
unclear whether the enzymatic restriction by EcoRI was not
rated into two different DNA oligonucleotides. For NarI
possible as a result of low thermal duplex stability of the
DNA oligonucleotides, different DNA–DNA duplexes and
modified double strands at ambient temperature or as a re-
DNA–RNA duplexes were formed, and thermal stabilities
sult of the homo-C-nucleoside modification. Interestingly,
and CD spectra were determined. Compared to NarI mis-
Ishiyama et al. observed a high resistance to different diges-
matched duplexes, homo-C-nucleoside-containing DNA–
tion enzymes of oligonucleotides containing homo-C-ade-
DNA duplexes showed a lowered melting temperature, sug-
nine.[2]
gesting a significant distortion of the double helix resulting
In addition, the circular dichroism spectra of all of the
in a lower hybrid stability. For thienopyrimidine- or benzo-
duplexes were recorded to investigate the duplex conforma-
diazepine-containing DNA oligonucleotides hybridized op-
tion. For NarI 19a, thienopyrimidine- and benzodiazepine-
posite an ABASIC site, the distortion was less significant
nucleoside-containing DNA-DNA duplexes overall B-type
than for a duplex formed between an unmodified oligonu-
was found, while DNA-RNA duplexes adopted A-type con-
cleotide and an oligonucleotide containing an ABASIC site.
formation. The same thing was also done for all of the
Both of the duplexes containing a “nonnatural” nucleoside
DNA–RNA hybrids. The shapes of the CD spectra were
wth an ABASIC site opposite the modification showed
less well pronounced, which indicates that the helices were
higher Tm values than the unmodified duplex containing an
less structured. For thienopyrimidine- or benzodiazepine-
ABASIC site. It remains to be seen whether this observa-
containing EcoRI-oligonucleotides 19b–19e, no conforma-
tion can be used beneficially in the application of artificial
tional difference relative to 19a was observed. In all cases,
nucleic acids, e.g., in therapeutic applications. UV melting
a positive Cotton effect was observed at 278 nm, and a
studies of the homo-C-nucleoside-containing RNA–DNA
negative effect, albeit less pronounced, at 250 nm. This is
duplexes revealed that their thermal stabilities were higher
typically found for B-type DNA (Figure 1).
than those of modified DNA–DNA duplexes, but that they
appeared to be lower than those of the reference RNA–
RNA duplex. The CD spectra of DNA–DNA and RNA–
DNA duplexes based on the homo-C-nucleoside-containing
oligonucleotides showed no difference compared to the un-
modified duplexes; the duplexes were found to be B-DNA
type for the DNA–DNA hybrids, and A-type for the RNA–
DNA hybrids. For the self-complementary EcoRI sequence,
a marked decrease in the Tm values was observed for homo-
C-nucleoside-containing oligonucleotides. Whether the re-
sistance to digestion is due to the homo-C-nucleoside or to
this low hybrid stability remains unclear.
In conclusion, the incorporation of a homo-C-nucleoside
into DNA decreased the thermal duplex stability towards a
complementary DNA or RNA strand. Whether this desta-
bilization of the artificial oligonucleotides is due to their
free rotation around the carbon–carbon bond axis must be
proved in future by structural modelling.
Experimental Section
General Methods: Melting points were determined with a Boetius
Figure 1. CD spectra of EcoRI oligonucleotides 19a–19e.
micro-heating plate BHMK 05 (Rapido, Dresden). Optical rota-
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6845

K. Höfler, T. Zimmermann, D. Peña Fuentes, C. Vogel, C. Meier
FULL PAPER
tions were measured with an automatic polarimeter GYROMAT 2-Amino-5-{[3,5-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-1,2-
(Dr. Kernchen Co.). 1H NMR spectra (250.13, 300.13, and
500.13 MHz) and 13C NMR spectra (62.9, 75.5, and 125.76 MHz)
were recorded with Bruker instruments AV 250, AV 300, and AV
500, respectively, with CDCl3 as solvent. The chemical shifts were
dideoxy-β-D-ribofuranos-1-yl]methyl}thiophene-3-carbonitrile (8):
Sulfur (77 mg, 2.4 mmol) and triethylamine (0.33 mL, 2.4 mmol)
were added to a stirred solution of compound 6 (745 mg, 1.6 mmol)
in dry N,N-dimethylformamide (8 mL). The mixture was stirred for
2 h at ambient temperature (monitored by TLC, solvent D), then
satd. aq. NaCl solution (90 mL) was added, and the aqueous layer
was extracted with CH2Cl2 (3ϫ170 mL). The combined organic
phases were washed with water (2 ϫ 90 mL), dried and concen-
trated. Column chromatography (solvent D) gave 8 (739 mg, 93%)
as a yellow syrup. [α]2D1 = –30.6 (c = 0.5, CHCl3). Rf = 0.22 (solvent
D). 1H NMR (300.13 MHz, CDCl3): δ = 0.96–1.08 [m, 28 H, 4
calibrated using solvent signals (CDCl3: δH = 7.26 ppm; δC
=
77.0 ppm). 1H and 13C NMR signals were assigned using DEPT
1
spectra, two-dimensional H,1H COSY and NOESY spectra, and
1H,13C correlation spectra (HMBC and HSQC). Washing solutions
were generally cooled to ca. 5 °C. The NaHCO3 solution was satu-
rated. Reactions were monitored by thin-layer chromatography
(TLC, Silica Gel 60, F254, Merck KGaA). The following solvent
3
3
2
systems (v/v) were used: n-hexane/ethyl acetate, 8:1 (A), 7:1 (B), 6:1 CH(CH3)2], 1.84 (dt, J1Ј,2Јa = J2Јa,3Ј = 7.93, J2Јa,2Јb = 12.84 Hz,
(C), 5:1 (D), 3:1 (E), 2:1 (F), 1:1 (G), and 1:2 (H); toluene/ethyl
acetate, 4:1 (I); ethyl acetate/methanol, 15:1 (J). The spots were
visualized by charring with H2SO4 (10% in ethanol). Flash
chromatography was carried out by elution from columns of slurry-
packed Silica Gel 60 (Merck, 63–200 μm). All solvents and reagents
were purified and dried according to standard procedures.[19] d-
Ribose was purchased from Alfa Aesar. Solutions in organic sol-
vents were dried with MgSO4, and concentrated under reduced
pressure (rotary evaporator).
1 H, 2Ј-Ha), 2.0 (ddd, 3J2Јb,3Ј = 4.53, 3J1Ј,2Јb = 6.5 Hz, 1 H, 2Ј-Hb),
2.80 (m, 1 H, 1ЈЈ-H), 3.70–3.78 (m, 2 H, 5Ј-Ha, 4Ј-H), 4.03 (m, 1
3
3
H, 5Ј-Hb), 4.22 (m, 1 H, 1Ј-H), 4.32 (dt, J2Јb,3Ј = 4.53, J2Јa,3Ј
=
3J3Ј,4Ј = 7.93 Hz, 1 H, 3Ј-H), 4.64 (br. s, 1 H, NH2), 6.44 (br. s, 1
H, 4-H) ppm. 13C NMR (74.47 MHz, CDCl3): δ = 12.52, 12.98,
13.35, 13.43 [4 CH(CH3)2], 16.93, 17.0, 17.09, 17.25, 17.39, 17.46,
17.55 [8 CH(CH3)2], 35.28 (C-1ЈЈ), 39.22 (C-2Ј), 63.78 (C-5Ј), 73.60
(C-3Ј), 76.89 (C-1Ј), 86.25 (C-4Ј), 87.36 (C-3), 115.53 (CN), 123.30
(C-4), 124.89 (C-5), 161.68 (C-2) ppm. HRMS (ESI-TOF+): calcd.
for C23H40N2O4SSi2Na [M + Na]+ 519.2140; found 519.2148.
C23H40N2O4SSi2 (496.81): calcd. C 55.61, H 8.12, N 5.64, S 6.45;
found C 55.35, H 7.99, N 5.38, S 6.41.
3-[3,5-O-(1,1,3,3-Tetraisopropyldisiloxan-1,3-diyl)-1,2-dideoxy-β-
D
-ribofuranos-1-yl]propanal (5): A solution of alcohol 4[17] (4.19 g,
10.0 mmol) in dry CH2Cl2 (25 mL) was added to a stirred solution
of 12-I-5-triacetoxyperiodinane (11.0 g, 26.0 mmol), NaHCO3
(1.81 g, 22.5 mmol), and molecular sieves (4 Å; ca. 5 g) in dry
CH2Cl2 (25 mL) at 0 °C under an Ar atmosphere. The mixture was
allowed to reach ambient temperature, and stirring was continued
for 6 h (monitored by TLC, solvent B). The mixture was then fil-
tered using a sintered glass filter funnel containing a layer of silica
gel, and the solids were washed with CH2Cl2 (2ϫ15 mL). The com-
bined filtrates were concentrated. Column chromatography (solvent
B) gave aldehyde 5 (3.50 g, 84%) as a colourless syrup. [α]2D2 = –24.2
(c = 1.0, CH2Cl2). Rf = 0.42 (solvent B). The NMR spectroscopic
data and combustion analysis were in perfect agreement with those
published by Wächtler et al.[15]
4-Amino-6-{[3,5-O-(1,1,3,3-tetraisopropyldisiloxan-1,3-diyl)-1,2-
dideoxy-β-D-ribofuranos-1-yl]methyl}thieno[2,3-d]pyrimidine (9): A
solution of compound 8 (994 mg, 2.0 mmol) in triethylorthofor-
mate (22 mL, 132 mmol) was heated under reflux for 2 h (moni-
tored by TLC, solvent G), and then the mixture was concentrated.
The resulting syrup was dissolved in a mixture of ethanol and
concd. aq. ammonia (1:1; 44 mL), and the mixture was heated un-
der reflux for 2 h (monitored by TLC, solvent G). The mixture was
allowed to reach ambient temperature, then it was concentrated.
Column chromatography (solvent G) gave compound 9 (932 mg,
89%) as colourless crystals. m.p. 154–156 °C (ethanol). [α]2D1
=
–11.4 (c = 0.5, CHCl3). Rf = 0.14 (solvent G). 1 H NMR
(300.13 MHz, CDCl3): δ = 0.94–1.06 [m, 28 H, 4 CH(CH3)2], 1.91
3
3
2
(dt, J1Ј,2Јa, = J2Јa,3Ј = 7.93, J2Јa,2Јb = 12.84 Hz, 1 H, 2Ј-Ha), 2.07
2-{3-[3,5-O-(Tetraisopropyldisiloxan-1,3-diyl)-1,2-dideoxy-β-D-
3
3
(ddd, J2Јb,3Ј = 4.34, J1Ј,2Јb = 6.61 Hz, 1 H, 2Ј-Hb), 3.08 (m, 1 H,
1ЈЈ-H), 3.71–3.81 (m, 2 H, 5Ј-Ha, 4Ј-H), 4.05 (m, 1 H, 5Ј-Hb), 4.32–
4.41 (m, 2 H, 1Ј-H, 3Ј-H), 5.40 (br. s, 2 H, NH), 6.90 (br. s, 1 H,
5-H), 8.41 (br. s, 1 H, 2-H) ppm. 13C NMR (74.47 MHz, CDCl3):
δ = 12.52, 12.93, 13.35, 13.42 [4 CH(CH3)2], 16.93, 16.99, 17.08,
17.24, 17.35, 17.38, 17.40, 17.56 [8 CH(CH3)2], 36.54 (C-1ЈЈ), 39.80
(C-2Ј), 63.87 (C-5Ј), 73.69 (C-3Ј), 76.96 (C-1Ј), 86.39 (C-4Ј), 115.61
(C-5), 116.37 (C-4a), 138.58 (C-7a), 153.36 (C-2) ppm. HRMS
(ESI-TOF+): calcd. for C24H42N3O4SSi2 [M + H]+ 524.2429; found
524.2426. C24H41N3O4SSi2 (523.84): calcd. C 55.03, H 7.89, N 8.02,
S 6.12; found C 55.17, H 7.92, N 8.21, S 6.05.
ribo-furanos-1-yl]propylidene}malononitrile (6): Malononitrile
(72.7 mg, 11.0 mmol) was added to a stirred solution of aldehyde
5 (1.25 g, 3.0 mmol) and aluminium oxide (1.38 g, 13.5 mmol; basic
activated, 90,101076 Merck) in dry toluene (100 mL). The mixture
was heated at reflux for 3 d (monitored by TLC, solvent C), then
the reaction mixture was cooled to ambient temperature, and the
insoluble solids were removed by filtration and washed with toluene
(3ϫ15 mL). The combined filtrates were concentrated, and the res-
idue was purified by column chromatography (solvent C) to give
Knoevenagel product 6 (906 mg, 65 %) as a pale yellow syrup.
[α]2D2 = –37.2 (c = 0.5, CHCl3). Rf = 0.28 (solvent C). 1H NMR
(250.13 MHz, CDCl3): δ = 0.97–1.11 [m, 28 H, 4 CH(CH3)2], 1.62– 4-N-Benzoyl-6-{[3,5-O-(1,1,3,3-tetraisopropyldisiloxan-1,3-diyl)-
1.90 (m, 3 H, 2Ј-Ha, 4-H), 2.07 (dd, 3J2Јb,3Ј = 4.53, 3J1Ј,2Јb = 6.6 Hz, 1,2-dideoxy-β-
-ribofuranos-1-yl]methyl}thieno[2,3-d]pyrimidine
3J2Јa,2Јb = 12.77 Hz, 1 H, 2Ј-Hb), 2.69 (m, 2 H, 3-H), 3.64–3.78 (m,
(10): Benzoyl chloride (0.35 mL, 3.0 mmol) was added to a solution
D
2 H, 4Ј-H, 5Ј-Ha), 3.96–4.13 (m, 2 H, 1Ј-H, 5Ј-Hb), 4.38 (dt, 3J2Јb,3Ј of compound 9 (1.05 g, 2.0 mmol) in dry pyridine (25 mL) at 0 °C.
= 4.6, 3J2Јa,3Ј = 3J3Ј,4Ј = 7.88 Hz, 1 H, 3Ј-H), 7.23 (t, 3J2,3 = 7.72 Hz, The mixture was stirred overnight at ambient temperature, then
1 H, 2-H) ppm. 13C NMR (62.90 MHz, CDCl3): δ = 12.52, 12.92, the excess benzoyl chloride was destroyed by addition of methanol
13.31, 13.46 [4 CH(CH3)2], 16.93, 17.01, 17.09, 17.24, 17.37, 17.41, (35 μL) at 0 °C. Stirring was continued for a further 30 min. The
17.52 [8 CH(CH3)2], 29.66 (C-3), 33.20 (C-4), 40.11 (C-2Ј), 63.40 mixture was poured into iced water (250 mL). The aqueous layer
(C-5Ј), 73.13 (C-3Ј), 73.37 (C-1Ј), 86.19 (C-4Ј), 89.48 (C-1), 110.53, was extracted with CH2Cl2 (3ϫ100 mL), and the combined or-
112.12 (2 CN), 114.21 [C(CH3)2], 169.91 (C-2) ppm. HRMS (ESI- ganic extracts were washed successively with NaHSO4 (15% aq.;
TOF+): calcd. for C23H41N2O4Si2 [M + H]+ 465.25990; found
465.26050. C23H40N2O4Si2 (464.75): calcd. C 59.44, H 8.68, N 6.03; (2ϫ70 mL). The organic phase was dried and concentrated. Col-
found C 59.65, H 8.45, N 6.06. umn chromatography (solvent I) gave 10 (1.04 g, 83%) as a yellow
3 ϫ 70 mL), iced water (70 mL), and satd. aq. NaHCO3
6846
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© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2015, 6841–6849

Synthesis of Homo-C-Nucleoside Phosphoramidites
foam. [α]2D1 = +13.3 (c = 1.0, CHCl3). Rf = 0.29 (solvent I). 1H
Ph), 8.84 (s, 1 H, 2-H), 11.30 (br. s, 1 H, NH) ppm. 13C NMR
NMR (500.13 MHz, CDCl3): δ = 0.95–1.09 [m, 28 H, 4 CH(CH3) (125.76 MHz, [D6]DMSO): δ = 36.29 (C-1ЈЈ), 39.71 (C-2Ј), 62.40
3
3
2
2], 1.93 (dt, J1Ј,2Јa, = J2Јa,3Ј = 7.93, J2Јa,2Јb = 12.84 Hz, 1 H, 2Ј- (C-5Ј), 72.04 (C-3Ј), 77.28 (C-1Ј), 87.72 (C-4Ј), 119.98 (C-5), 122.75
3
3
Ha), 2.07 (ddd, J2Јb,3Ј = 4.06, J1Ј,2Јb = 6.48 Hz, 1 H, 2Ј-Hb), 3.16
(C-4a), 128.45, 128.53 (m, o-Ph), 132.53 (p-Ph), 133.25 (ipso-Ph),
3
(m, 1 H, 1ЈЈ-H), 3.73–3.81 (m, 2 H, 5Ј-Ha, 4Ј-H), 4.07 (dd, J4Ј,5Јb 140.68 (C-6), 152.13 (C-2, C-7a), 166.25 (C=O), 169.31 (C-4) ppm.
= 2.90, J5Јa,5Јb = 10.73 Hz, 1 H, 5Ј-Hb), 4.36 (dt, J3Ј,4Ј = 4.38 Hz, HRMS (ESI-TOF+): calcd. for C19H20N3O4S [M + H]+ 386.1169;
3
3
1 H, 3Ј-H), 4.40 (m, 1 H, 1Ј-H), 7.37 (s, 1 H, 5-H), 7.53 (m, 2 H, found 386.1172. C19H19N3O4S (385.44): calcd. C 59.21, H 4.97,
m-Ph), 7.60–7.65 (m, 1 H, p-Ph), 7.98 (m, 2 H, o-Ph), 8.69 (s, 1 H,
2-H), 8.87 (s, 1 H, NH) ppm. 13C NMR (125.76 MHz, CDCl3): δ
= 12.54, 12.97, 13.36, 13.42 [4 CH(CH3)2], 16.94, 17.0, 17.08, 17.24,
17.37, 17.38, 17.42, 17.54 [8 CH(CH3)2], 36.80 (C-1ЈЈ), 39.77 (C-
2Ј), 64.09 (C-5Ј), 74.04 (C-3Ј), 76.85 (C-1Ј), 86.52 (C-4Ј), 121.07
(C-5), 121.66 (C-4a), 127.79 (o-Ph), 129.0 (m-Ph), 133.03 (p-Ph),
133.21 (ipso-Ph), 140.04 (C-6), 150.86 (C-7a), 151.91 (C-2), 165.71
(C=O), 171.43 (C-4) ppm. HRMS (ESI-TOF+): calcd. for
C3 1 H4 6 N3 O5 SSi2 [M + H]+ 628.2691; found 628.2694.
C31H45N3O5SSi2 (627.95): calcd. C 59.29, H 7.22, N 6.69, S 5.11;
found C 59.44, H 7.17, N 6.55, S 5.00.
N10.90, S 8.32; found C 59.30, H 5.07, N 10.73, S 8.50.
4-[2-(1,2-Dideoxy-β-D-ribofuranos-1-yl)ethyl]2-phenyl-3H-1,5-benzo-
diazepine (15): Yellow foam (308 mg, 94%). [α]2D3 = –13.2 (c = 1.1,
1
CHCl3). Rf = 0.29 (solvent J). H NMR (300.13 MHz, CDCl3): δ
3
3
2
= 1.69 (ddd, J2Јa,3Ј = 6.40, J1Ј,2Јa = 9.63 Hz, J2Јa,2Јb = 13.22 Hz,
1 H, 2Ј-Ha), 1.80–2.03 (m, 3 H, 2ЈЈ-H, H- 2Ј-Hb), 2.64 (m, 3 H,
1ЈЈ-H, O5Ј-H), 2.82 (br. s, 1 H, O3Ј-H), 3.25 (br. s, 2 H, 3-H), 3.51
3
2
(dd, J4Ј,5Јa
=
3JOH-5Ј,5Јa = 4.52, J5Јa,5Јb = 11.70 Hz, 1 H, 5Ј-Ha),
3
3.61 (dd, J4Ј,5Јb = 3JOH-5Ј,5Јb = 3.97 Hz, 1 H, 5Ј-Hb), 3.75 (m, 1 H,
4Ј-H), 4.09–4.18 (m, 1 H, 1Ј-H), 4.23 (dt, 3J2Јb,3Ј = 3JOH-3Ј,3Ј = 2.59,
3J2Јa,3Ј = 6.40 Hz, 1 H, 3Ј-H), 7.24–7.32, 7.42–7.57 (2 m, 7 H, m-,
p-Ph, 6-H, 7-H, 8-H, 9-H), 8.00–8.06 (o-Ph) ppm. 13C NMR
(75.47 MHz, CDCl3): δ = 31.27 (C-2ЈЈ), 36.33 (C-1ЈЈ), 37.84 (C-3),
41.08 (C-2Ј), 63.05 (C-5Ј), 73.46 (C-3Ј), 77.69 (C-1Ј), 86.93 (C-4Ј),
125.18, 125.31, 127.60, 128.51, 128.78, 130.82 (m-, p-Ph, C-6, C-7,
C-8, C-9), 128.22 (o-Ph), 137.0 (ipso-Ph), 140.03, 140.62 (C-5a, C-
9a), 153.85 (C-4), 161.11 (C-2) ppm. HRMS (ESI-TOF+): calcd.
for C22 H2 5N2O3 [M + H]+ 365.18597; found 365.18554.
C22H24N2O3 (364.45): calcd. C 72.51, H 6.64, N 7.69; found C
71.75, H 6.66, N 7.42.
4-{2-[3,5-(1,1,3,3-Tetraisopropyldisiloxane-1,3-diyl)-1,2-dideoxy-β-
D
-ribofuranos-1-yl]ethyl}2-phenyl-3H-1,5-benzodiazepine (14): o-
Phenylenediamine (216 mg, 2.0 mmol) was added to a solution of
compound 7[15] (1.03 g, 2.0 mmol) in ethanol (200 mL). The mix-
ture was heated under reflux for 5 h (monitored by TLC, solvent
A), then it was allowed to reach ambient temperature, and then
concentrated. Column chromatography (solvent A) gave 14
(862 mg, 71%) as a yellow syrup. [α]2D2 = –36.0 (c = 1.0, CHCl3).
1
Rf = 0.17 (solvent A). H NMR (300.13 MHz, CDCl3): δ = 0.96–
Introduction of the Dimethoxytrityl Protecting Group: 4,4Ј-Dimeth-
oxytrityl chloride (847 mg, 2.5 mmol), 4-(dimethylamino)pyridine
(24 mg, 0.2 mmol), and triethylamine (0.5 mL, 3.6 mmol) were
added to a stirred solution of 11 (771 mg, 2.0 mmol) or 15 (729 mg,
2.0 mmol) in dry N,N-dimethylformamide (18 mL) at ambient tem-
perature. After 24 h, an additional amount of 4,4Ј-dimethoxytrityl
chloride (169 mg, 0.5 mmol) was added, and stirring was continued
for a further 24 h. Methanol (24 μL) was added, and after 30 min,
the mixture was poured into iced water (70 mL). The aqueous layer
was extracted with CH2Cl2 (3ϫ120 mL), and the combined or-
ganic extracts were dried and concentrated. The residue was puri-
fied by column chromatography (solvent G).
1.12 [m, 28 H, 4 CH(CH3)2], 1.76–2.06 (m, 4 H, 2ЈЈ-H, 2Ј-H), 2.55–
2.78 (m, 2 H, 1ЈЈ-H), 3.29 (br. s, 2 H, 3-H), 3.68–3.76 (m, 2 H, 4Ј-
H, 5Ј-Ha), 3.98–4.14 (m, 2 H, 5Ј-Hb, 1Ј-H), 4.38 (dt, 3J2Јb,3Ј = 4.34,
3J2Јa,3Ј = 7.93 Hz, 1 H, 3Ј-H), 7.27–7.33, 7.43–7.58 (m, 7 H, m-, p-
Ph, 6-H, 7-H, 8-H, 9-H), 8.04–8.09 (m, 2 H, o-Ph) ppm. 13C NMR
(75.47 MHz, CDCl3): δ = 12.54, 12.93, 13.35, 13.47 [4 CH(CH3)2],
16.96, 17.04, 17.12, 17.28, 17.41, 17.42, 17.55 [8 CH(CH3)2], 31.8
(C-2ЈЈ), 36.3 (C-1ЈЈ), 37.89 (C-3), 40.32 (C-2Ј), 63.89 (C-5Ј), 73.63
(C-3Ј), 76.83 (C-1Ј), 85.92 (C-4Ј), 124.96, 125.13, 127.74, 128.50,
128.75, 130.69 (m-, p-Ph, C-6, C-7, C-8, C-9), 128.22 (o-Ph), 137.11
(ipso-Ph), 140.43 140.60 (C-5a, C-9a), 153.70 (C-2), 160.51 (C-4)
ppm. HRMS (ESI-TOF+): calcd. for C34H50N2O4Si2Na [M +
Na]+ 629.32013; found 629.32013. C34H50N2O4Si2 (606.95): calcd. 4-N-Benzoyl-6-[(5-O-dimethoxytrityl-1,2-dideoxy-β-
D
-ribofuranos-
1-yl)methyl]thieno[2,3-d]pyrimidine (12): Pale yellow foam (1.14 g,
83%). [α]2D1 = +12.7 (c = 1.0, CHCl3). Rf = 0.29 (solvent H). 1H
C 67.28, H 8.30, N 4.62; found C 66.99, H 8.11, N 4.68.
Removal of the Tetraisopropyldisiloxane Protecting Group: A solu-
tion of tetrabutylammonium fluoride trihydrate (788 mg,
2.5 mmol) in acetone (0.5 mL) was added dropwise to a solution
of compound 10 (628 mg, 1.0 mmol) or 14 (607 mg, 1.0 mmol) in
acetone (1.5 mL). The reaction mixture was stirred at ambient tem-
perature for 1.5 h (monitored by TLC, solvent D), and then con-
centrated. The residue was purified by column chromatography
(solvent J).
3
3
NMR (300.13 MHz, CHCl3): δ = 1.91 (ddd, J1Ј,2Јa, = 9.3, J2Јa,3Ј
2
3
= 6.0, J2Јa,2Јb = 13.1 Hz, 1 H, 2Ј-Ha), 1.99 (ddd, J2Јb,3Ј = 2.64,
3J1Ј,2Јb = 7.20 Hz, 1 H, 2Ј-Hb), 3.12 (dd, J4Ј,5Јa = 5.8, J5Јa,5Јb
=
=
3
2
3
9.7 Hz, 1 H, 5Ј-Ha), 3.21 (m, 2 H, 1ЈЈ-H), 3.27 (dd, J4Ј,5Јb
4.47 Hz, 1 H, 5Ј-Hb), 3.77 (2 s, 6 H, CH3-O), 3.94 (m, 1 H, 4Ј-H),
3
4.31 (dt, J3Ј,4Ј = 6.04 Hz, 1 H, 3Ј-H), 4.50 (m, 1 H, 1Ј-H), 6.79–
6.82, 7.14–7.24, 7.28–7.33, 7.40–7.44 (4 m, 13 H, Ar-DMTr), 7.38
(s, 1 H, 5-H), 7.50–7.55 (m, 2 H, m-Ph-Bz), 7.60–7.65 (m, 1 H, p-
Ph-Bz), 7.96 (m, 2 H, o-Ph-Bz), 8.72 (m, 2 H, 2-H, NH) ppm. 13C
NMR (75.47 MHz, CHCl3): δ = 37.12 (C-1ЈЈ), 40.1 1 (C-2Ј), 55.19
(2 CH3-O), 64.41 (C-5Ј), 74.48 (C-3Ј), 77.83 (C-1Ј), 86.10 (C-4Ј),
86.19 [C(Ph)3], 113.12, 126.76 (Ar-DMTr), 120.82 (C-5), 121.71 (C-
4a), 127.81 (o-Ph-Bz, Ar-DMTr), 128.12, 130.01, 130.03, 135.90,
135.99, 144.76, 158.44 (Ar-DMTr), 128.97 (m-Ph-Bz), 132.99 (p-
Ph-Bz), 133.14 (ipso-Ph-Bz), 140.47 (C-6), 150.79 (C-7a), 151.87
(C-2), 165.61 (C=O), 171.32 (C-4) ppm. HRMS (ESI-TOF+):
calcd. for C40H38N3O6S [M + H]+ 688.24758; found 688.24849.
C40H37N3O6S (687.81): calcd. C 69.84, H 5.42, N 6.11, S 4.66;
found C 70.01, H 5.34, N 6.05, S 4.78.
4-N-Benzoyl-6-[(1,2-dideoxy-β-D-ribofuranos-1-yl)methyl]thieno-
[2,3-d]pyrimidine (11): Colourless crystals (366 mg, 95%). m.p. 152–
154 °C (ethanol). [α]2D1 = +15.9 (c = 0.5, MeOH). Rf = 0.26 (solvent
1
3
J). H NMR (500.13 MHz, [D6]DMSO): δ = 1.68 (ddd, J1Ј,2Јa, =
3
2
9.78, J2Јa,3Ј = 5.99, J2Јa,2Јb = 12.72 Hz, 1 H, 2Ј-Ha), 1.82 (ddd,
3J2Јb,3Ј = 1.87, J1Ј,2Јb = 5.40 Hz, 1 H, 2Ј-Hb), 3.11 (dd, J1ЈЈa,1Ј
=
=
3
3
2
3
6.74, J1ЈЈa,1ЈЈb = 14.87 Hz, 1 H, 1ЈЈ-Ha), 3.17 (dd, J1ЈЈb,1Ј
4.76 Hz, 1 H, 1ЈЈ-Hb), 3.30 (dt, J4Ј,5Јa = JOH-5Ј,5Јa = 6.74, J5Јa,5Јb
= 11.35 Hz, 2 H, 5Ј-Ha), 3.39 (dt, J4Ј,5Јb = JOH-5Ј,5Јb = 5.49 Hz, 1
H, 5Ј-Hb), 3.67 (td, J3Ј,4Ј = 2.52 Hz, 1 H, 4Ј-H), 4.05 (m, 1 H, 3Ј-
3
3
2
3
3
3
3
H), 4.30 (m, 1 H, 1Ј-H), 4.63 (t, JOH-5Ј,5Јb = 5.53 Hz, 1 H, O5Ј-
3
H), 4.92 (d, JOH-3Ј,3Ј = 4.10 Hz, 1 H, O3Ј-H), 7.18 (s, 1 H, 5-H), 4-[2-(5-O-Dimethoxytrityl-1,2-dideoxy-β-
D
-ribofuranos-1-yl)ethyl]-
7.56 (m, 2 H, m-Ph), 7.64–7.67 (m, 1 H, p-Ph), 8.07 (m, 2 H, o-
2-phenyl-3H-1,5-benzodiazepine (16): Pale yellow foam (867 mg,
Eur. J. Org. Chem. 2015, 6841–6849
© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
6847

K. Höfler, T. Zimmermann, D. Peña Fuentes, C. Vogel, C. Meier
FULL PAPER
65%). [α]2D3 = –13.5 (c = 1.0, CHCl3). Rf = 0.18 (solvent G). 1H
1,5-benzodiazepine (17): Yellow foam (659 mg, 76%). Rf = 0.23/0.29
3
3
1
NMR (300.13 MHz, CHCl3): δ = 1.71 (ddd, J2Јa,3Ј = 6.35, J1Ј,2Јa (solvent E; two diastereomers). H NMR (300.13 MHz, CHCl3): δ
= 9.47 Hz, 2J2Јa,2Јb = 13.06 Hz, 1 H, 2Ј-Ha), 1.84–2.02 (m, 4 H, 2ЈЈ-
= 1.06, 1.08, 1.11, 1.13, 1.14, 1.16, 1.17, 1.18 [8 s, 12 H, 2 CH(CH3)
3
3
H, O3Ј-H, 2Ј-Hb), 2.59 (ddd, J1ЈЈa,2ЈЈa = 6.09, J1ЈЈa,2ЈЈb = 9.03 Hz, 2], 1.65–1.76 (m, 1 H, 2Ј-Ha), 1.92–2.11 (m, 3 H, 2ЈЈ-H, 2Ј-Ha),
3
3J1ЈЈa,1ЈЈb = 15.83 Hz, 1 H, 1ЈЈ-Ha), 2.72 (ddd, J1ЈЈb,2ЈЈb = 6.09,
2.43, 2.56 (2 t, 3JCH2-OP,CH2CN = 6.42 Hz, 2 H, CH2CN), 2.63–2.68,
2.72–2.84 (2 m, 2 H, 1ЈЈ-H), 3.07–3.12 (m, 2 H, 5Ј-H), 3.50-3.61
[m, 2 H, CH(CH3)2], 3.63–3.75 (m, 2 H, CH2-OP), 3.76, 3.77 (2 s,
3
2
3J1ЈЈb,2ЈЈa = 9.03 Hz, 1 H, 1ЈЈ-Hb), 3.04 (dd, J4Ј,5Јa = 6.18, J5Јa,5Јb
3
= 9.65 Hz, 1 H, 5Ј-Ha), 3.23 (br. s, 2 H, 3-H), 3.13 (dd, J4Ј,5Јb
=
4.72 Hz, 1 H, 5Ј-Hb), 3.77 (2 s, 6 H, CH3-O), 3.86 (m, 1 H, 4Ј-H), 6 H, CH3-O), 4.03–4.09 (m, 1 H, 4Ј-H), 4.11–4.22 (m, 1 H, 1Ј-H),
4.09–4.20 (m, 1 H, 1Ј-H), 4.26 (m, 1 H, 3Ј-H), 6.78–6.84, 7.17–7.25, 4.47–4.43 (m, 1 H, 3Ј-H), 6.77–6.83, 7.18–7.29, 7.31–7.35, 7.39–
7.27–7.33, 7.51–7.55 (4 m, 20 H, m-, p-Ph, Ar-DMTr, 6-H, 7-H, 8-
7.47, 7.52–7.55 (5 m, 20 H, m-, p-Ph, Ar-DMTr, 6-H, 7-H, 8-H, 9-
H, 9-H), 8.0–8.03 (m, 1 H, o-Ph) ppm. 13C NMR (75.47 MHz, H), 8.01–8.04 (m, 2 H, o-Ph) ppm. 13C NMR (75.47 MHz, CHCl3):
CHCl3): δ = 31.95 (C-2ЈЈ), 36.66 (C-1ЈЈ), 37.94 (C-3), 40.55 (C-2Ј), δ = 20.09, 20.15, 20. 27, 20.32 (CH2CN), 24.37, 24.46, 24.51, 24.52,
55.17 (2 CH3-O), 64.72 (C-5Ј), 74.70 (C-3Ј), 77.61 (C-1Ј), 85.33 (C- 24.60, 24.62 [CH(CH3)2], 31.69, 31.75 (C-2ЈЈ), 36.76 (C-1ЈЈ), 37.93
4Ј), 86.11 [C(Ph)3], 113.09, 124.99, 125.15, 126.76, 127.79, 128.15,
(C-3), 40.0, 40.05 (C-2Ј), 43.03, 43.07, 43.19, 43.23 [CH(CH3)2],
128.49, 128.74, 130.02, 130.04, 130.69, 136.01, 137.02, 144.81, 55.16 (CH3-O), 58.04, 58.12, 58.29, 58.36 (CH2-OP), 64.26, 64.33
158.46 (ipso-, m-, o-, p-Ph, Ar-DMTr, C-6, C-7, C-8, C-9), 140.40, (C-5Ј), 75.59, 75.82, 75.98, 76.21 (C-3Ј), 77.90, 77.96 (C-1Ј), 85.13,
140.59 (C-5a, C-9a), 153.81 (C-2), 160.55 (C-4) ppm. HRMS (ESI-
85.20, 85.39, 85.45 (C-4Ј), 85.92 [C(Ph)3], 113.0 (Ar-DMTr),
TOF+): calcd. for C43H43N2O5 [M + H]+ 667.31665; found 117.47, 117.54 (CN), 124.95, 125.11, 126.66, 126.69, 127.69, 127.71,
667.31698. C43H42N2O5 (666.82): calcd. C 77.45, H 6.35, N 4.20;
found C 77.59, H 6.44, N 4.21.
127.79, 128.14, 128.22, 128.26, 128.50, 128.73, 130.06, 130.09,
130.013, 130.67, 136.06, 136.13, 136.16, 137.02, 144.89, 158.38,
158.41 (ipso-, m-, o-, p-Ph, Ar-DMTr, C-6, C-7, C-8, C-9), 140.05,
140.59 (C-5a, C-9a), 153.70, 153.74 (C-2), 160.61, 160.64 (C-4)
ppm. 31P NMR (121.49 MHz): δ = 147.59, 147.91 ppm. MS (EI):
m/z (%) = 866 (1.38) [M]+. C52H59N4O6P (867.04): calcd. C 72.03,
H 6.86, N 6.46; found C 71.86, H 6.88, N 6.53.
Introduction of the 2-(Cyanoethoxy)(diisopropylamino)phosphine
Group: Diisopropylethylamine (0.4 mL, 2.4 mmol) and 2-cyanoe-
thyl-N,N-diisopropyl chlorophosphoramidite (290 μL, 1.3 mmol)
were added to a stirred solution of 12 (687 mg, 1.0 mmol) or 15
(667 mg, 1.0 mmol) in dry CH2Cl2 (19 mL) at ambient temperature.
After 6 h (monitored by TLC, solvent G), methanol (27 μL) was
added, and stirring was continued for 30 min. The mixture was
diluted with CH2Cl2 (150 mL), washed with satd. aq. NaHCO3
(60 mL), dried, and concentrated. The residue was purified by col-
umn chromatography (solvent F).
Synthesis of the DNA Oligonucleotides: DNA oligonucleotides were
synthesized on a 1 μmol scale using Bz-protected dA, Bz-protected
dC, dmf (dimethylformamidine)-protected dG, and T phosphor-
amidites (Glen Research), using an H-8 DNA/RNA/LNA synthe-
sizer (K&A Laborgeräte) with CPG (controlled pore glass) solid
supports purchased from Biosearch Technologies. For the incorpo-
ration of the modified phosphoramidites, the coupling was re-
peated once, with each run lasting 199 s. DNA oligonucleotides
were purified by reverse-phase HPLC using a C-18 column
(Merck), using a 50 min linear gradient of acetonitrile, 0–20%, in
triethylammonium acetate buffer, pH 6.9, at room temperature
(Table 6).
4-N-Benzoyl-6-({5-O-dimethoxytrityl-3-O-[(2-cyanoethoxy)(diiso-
propylamino)phosphanyl]-1,2-dideoxy-β-D-ribofuranos-1-yl}meth-
yl)thieno-[2,3-d]pyrimidine (13): Colourless foam (710 mg, 80%). Rf
= 0.25/0.28 (solvent G; two diastereomers). 1 H NMR
(500.13 MHz, CHCl3): δ = 1.04, 1.05, 1.10, 1.12, 1.14, 1.15, 1.16 [8
s, 12 H, 2 CH(CH3)2], 1.91, 2.14 (2 m, 2 H, 2Ј-H), 2.42, 2.57 (2 t,
3JCH2-OP,CH2CN = 6.40 Hz, 2 H, CH2CN), 3.14 (m, 1 H, 5Ј-Ha),
3
3
3.17 (dd, J4Ј,5Јb = 4.41, J5Јa,5Јb = 10.36 Hz, 1 H, 5Ј-Hb), 3.26 (m,
2 H, 1ЈЈ-H), 3.53 [m, 2 H, CH(CH3)2], 3.62–3.74, 3.79–3.82 (m, 2
H, CH2-OP), 3.77 (3 s, 6 H, CH3-O), 4.12 (m, 1 H, 4Ј-H), 4.42 (m,
Table 6. MALDI-TOF mass data (m/z) for DNA oligonucleotides.
Compound
Calcd.
Found
1 H, 3Ј-H), 4.50 (m, 1 H, 1Ј-H), 6.79–6.82, 7.14–7.18, 7.24–7.28,
18
3580.6
3642.6
3656.6
3656.6
3739.7
3739.7
3709.7
3749.7
3733.7
3724.7
3600.6
3677.7
3431.6
3584.7
3644.0
3655.7
3655.9
3738.5
3738.6
3711.4
3751.5
3735.4
3726.2
3599.9
3677.3
3433.6
4
7.30–7.34, 7.44–7.46 (5 m, 13 H, Ar-DMTr), 7.40 (d, J1ЈЈ,5
=
19a
19b
19c
19d
19e
21a
21b
21c
21d
21e
23
4.73 Hz, 1 H, 5-H), 7.50–7.53 (m, 2 H, m-Ph-Bz), 7.61–7.63 (m, 1
H, p-Ph-Bz), 7.96 (m, 2 H, o-Ph-Bz), 8.70 (s, 1 H, 2-H), 8.83 (br.
s, 1 H, NH) ppm. 13C NMR (125.76 MHz, CHCl3): δ = 20.09,
20.15, 20. 27, 20.32 (CH2CN), 24.36, 24.39, 24.43, 24.50, 24.52,
24.56, 24.57 [CH(CH3)2], 36.99, 37.02 (C-1ЈЈ), 39.62, 39.66, 39.68
(C-2Ј), 43.07, 43.11, 43.16, 43.21 [CH(CH3)2], 55.15, 55.17 (CH3-
O), 58.11, 58.16, 58.26, 58.31 (CH2-OP), 63.96, 64.04 (C-5Ј), 75.40,
75.53, 75.77, 75.91 (C-3Ј), 78.12, 78.21 (C-1Ј), 85.64, 85.69, 85.93,
85.97 (C-4Ј), 86.0, 86.01 [C(Ph)3], 113.04 (Ar-DMTr), 117.41,
117.51 (CN), 126.66, 126.69, 127.71, 127.73, 128.19, 128.23, 130.02,
130.05, 130.07, 130.11, 135.95, 136.10, 136.13, 144.82, 158.39,
158.41 (Ar-DMTr), 120.64, 120.75 (C-5), 121.80, 121.82 (C-4a),
127.83 (o-Ph-Bz), 128.92 (m-Ph-Bz), 132.91 (p-Ph-Bz), 133.23,
133.27 (ipso-Ph-Bz), 140.65, 140.70 (C-6), 150.87, 150.89 (C-7a),
151.86, 151.89 (C-2), 165.64 (C=O), 171.09 (C-4) ppm. 31P NMR
(121.49 MHz, CHCl3): δ = 147.82, 147.94 ppm. HRMS (ESI-
TOF+): calcd. for C49H54N5O7PSNa [M + Na]+ 910.33738; found
910.33638. C49H54N5O7PS (888.03): calcd. C 66.27, H 6.13, N 7.89,
S 3.61; found C 66.17, H 6.21, N 7.77, S 3.86.
25
Synthesis of the RNA Oligonucleotides: RNA was synthesized on a
0.5 μmol scale using polystyrene (PS) 2Ј-O-acetyl-protected RNA
Custom Primer Support from GE Healthcare, and 2Ј-TC protected
RNA phosphoramidites (SAFC) used as recommended by the
manufacturer’s protocol. For RNA synthesis, each coupling was
repeated three times, each for 120 s. After the synthesis, the solid
support was washed with anhydrous acetonitrile, and dried with
argon. The RNA was deprotected as follows: it was treated with
anhydrous ethylenediamine for 2 h at room temperature, followed
4-(2-{5-O-Dimethoxytrityl-3-O-[(2-cyanoethoxy)(diisopropylamino)-
phosphanyl]-1,2-dideoxy-β-
D-ribofuranos-1-yl}ethyl)2-phenyl-3H-
6848
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© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2015, 6841–6849

Synthesis of Homo-C-Nucleoside Phosphoramidites
by flushing with nitrogen, elution with buffer (100 mm triethyl-
amine, pH 7.0), filtration, and freeze-drying. The crude RNA oli-
gonucleotides were purified by anion-exchange HPLC using a
DNAPac PA-200 column (Thermo Scientific) with a linear gradient
of 0.1 m NaClO4 buffer (pH = 8.0 in 0.025 m Tris-HCl) in 10%
acetonitrile starting from 0% buffer to 100% over 40 min at 50 °C.
The strands were desalted by size-exclusion chromatography on an
XK16/40 column custom-packed with Sephadex G-25 (GE
Healthcare), eluting with sterile water (Table 7).
a fellowship from the Deutscher Akademiker Austauschdienst
(DAAD).
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20
24
3887.6
3744.5
3887.4
3744.6
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phosphate buffer, 140 mm NaCl, 1 mm EDTA, pH 6.8). The UV
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Received: July 29, 2015
Supporting Information (see footnote on the first page of this arti-
cle): HPLC profiles and MALDI-TOF spectra of oligonucleotides
18, 19b–19e, 21b, 21e, and 22–24.
Acknowledgments
The authors are grateful for financial support from the University
of Hamburg and the University of Rostock. D. P. F. is grateful for
Published Online: September 29, 2015
Eur. J. Org. Chem. 2015, 6841–6849
© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
6849