Watson-Crick base-pairing properties of tricyclo-DNA

Article abstract of DOI:10.1021/ja025569+

Tricyclo-DNA belongs to the family of conformationally restricted oligodeoxynucleotide analogues. It differs structurally from DNA by an additional ethylene bridge between the centers C(3′) and C(5′) of the nucleosides, to which a cyclopropane unit is fus

Full text of DOI:10.1021/ja025569+

Published on Web 05/07/2002
Watson-Crick Base-Pairing Properties of Tricyclo-DNA
Dorte Renneberg and Christian J. Leumann*
Contribution from the Department of Chemistry and Biochemistry, UniVersity of Bern,
Freiestrasse 3, CH-3012 Bern, Switzerland
Received January 11, 2002
Abstract: Tricyclo-DNA belongs to the family of conformationally restricted oligodeoxynucleotide analogues.
It differs structurally from DNA by an additional ethylene bridge between the centers C(3′) and C(5′) of the
nucleosides, to which a cyclopropane unit is fused for further enhancement of structural rigidity. The synthesis
of the hitherto unknown tricyclodeoxynucleosides containing the bases cytosine and guanine and of the
corresponding phosphoramidite building blocks is described, as well as a structural description of a
representative of an R- and a â-tricyclodeoxynucleoside by X-ray analysis. Tricyclodeoxynucleoside building
blocks of all four bases were used for the synthesis of fully modified mixed-base oligonucleotides. Their
Watson-Crick pairing properties with complementary DNA, RNA, and with itself were investigated by UV
melting curves, CD spectroscopy, and molecular modeling. Tricyclo-DNA was found to be a very stable
Watson-Crick base-pairing system. A UV melting curve analysis of the decamers tcd(pcgtgacagtt) and
tcd(paactgtcacg) showed increased thermal stabilities of up to ∆Tm/mod. ) +1.2 °C with complementary
DNA and +2.4 °C with complementary RNA. With itself, tricyclo-DNA showed an increase in stability of
+3.1 °C/base pair relative to DNA. Investigations into the thermodynamic properties of these decamers
revealed an entropic stabilization and an enthalpic destabilization for the tricyclo-DNA/DNA duplexes. CD
spectroscopic structural investigations indicated that tricyclo-DNA containing duplexes preferrably exist in
an A-conformation, a fact which is in agreement with results from molecular modeling.
Introduction
DNA analogues are of interest in antisense technology to
selectively inhibit the gene expression on the level of transla-
tion.1,2 Various mechanisms of inhibition are known, the most
important ones being steric blockage or RNase H-induced
degradation of the mRNA after duplex formation.3,4 Several
antisense oligonucleotides of the first and second generation
are currently being evaluated in clinical trials for the treatment
of diseases, for example, various forms of cancer5 and Crohn’s
disease.6 These DNA analogues are typically oligodeoxynucleo-
tide phosphorothioates or 2′-O-alkyl-ribooligonucleotides. In
1998, the first antisense drug, the oligodeoxynucleotide phos-
phorothioate Vitravene (ISIS-2922), became available on the
market for use in the treatment of cytomegalovirus (CMV)-
induced retinitis. Besides their use in therapy, oligonucleotide
analogues also play an increasingly important role in the
systematic and high-throughput screening of gene function, and
in drug target validation.7
Figure 1. Chemical structures of bicyclo-DNA and tricyclo-DNA.
single strands as a means to increase duplex stability has been
evaluated within the past decade and has led to DNA analogues
with unprecedented thermal duplex stabilities and improved
biostability.8 The locked nucleic acids (LNA)9-11 and the hexitol
nucleic acids (HNA)12,13 are notable members of this family
which show strongly enhanced binding properties to DNA and
RNA. The pioneering contribution from our laboratory in this
field was the analogue bicyclo-DNA (Figure 1).14 Homopurine
In the context of improving the binding properties of antisense
oligonucleotides, the concept of conformational restriction of
(8) Leumann, C. J. Bioorg. Med. Chem. 2002, 10, 841-854.
(9) Koshkin, A. A.; Singh, S. K.; Nielsen, P.; Rajwanshi, V. K.; Kumar, R.;
Meldgaard, M.; Olsen, C. E.; Wengel, J. Tetrahedron 1998, 54, 3607-
3630.
* To whom correspondence should be addressed. Phone: +41-31-631-
4355. Fax: +41-31-631-3422. E-mail: leumann@ioc.unibe.ch.
(1) Myers, K. J.; Dean, N. M. Trends Pharmacol. Sci. 2000, 21, 19-23.
(2) Uhlmann, E. Curr. Opin. Drug DiscoVery DeV. 2000, 3, 203-213.
(3) Baker, B. F.; Monia, B. P. Biochim. Biophys. Acta 1999, 1489, 3-18.
(4) Crooke, S. T. Biochim. Biophys. Acta 1999, 1489, 31-44.
(5) Flanagan, W. M. Cancer Metastatis ReV. 1998, 17, 169-176.
(6) Crooke, S. T. Antisense Nucleic Acid Drug DeV. 1998, 8, 115-122.
(7) Taylor, M. F.; Wiederholt, K.; Sverdrup, F. Drug DiscoVery Today 1999,
4, 562-567.
(10) Singh, S. K.; Nielsen, P.; Koshkin, A. A.; Wengel, J. Chem. Commun.
1998, 455-456.
(11) Obika, S.; Nanbu, D.; Hari, Y.; Andoh, J.-I.; Morio, K.-I.; Doi, T.; Imanishi,
T. Tetrahedron Lett. 1998, 39, 5401-5404.
(12) Hendrix, C.; Rosemeyer, H.; Verheggen, I.; Seela, F.; Van Aerschot, A.;
Herdewijn, P. Chem.-Eur. J. 1997, 3, 110-120.
(13) Van Aerschot, A.; Verheggen, I.; Hendrix, C.; Herdewijn, P. Angew. Chem.,
Int. Ed. Engl. 1995, 34, 1338-1339.
(14) Tarko¨y, M.; Bolli, M.; Leumann, C. HelV. Chim. Acta 1994, 77, 716-
744.
9
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J. AM. CHEM. SOC. 2002, 124, 5993-6002
5993

A R T I C L E S
Renneberg and Leumann
Scheme 1a
bicyclo-DNA sequences were shown to form entropically
stabilized duplexes with a preference for the Hoogsteen and/or
reverse Hoogsteen association mode. This change in association
mode relative to DNA was identified to be the consequence of
an alteration of the backbone torsion angle γ in the bicyclo-
DNA backbone.15 With mixed-base bicyclo-DNA sequences,
Watson-Crick duplex formation with DNA or RNA occurred
readily with thermal stabilities near that of DNA duplexes.16
Tricyclo-DNA (Figure 1) is a second generation derivative
of bicyclo-DNA. The additional cyclopropane ring was designed
to confer further conformational stabilization to the nucleoside
and eventually a partial correction of the torsion angle γ from
the anticlinal range toward the gauche range. Furthermore, the
increased hydrophobicity of the tricyclo-DNA backbone is
expected to help cellular uptake. We showed previously that
homobasic adenine- and thymine-containing tricyclo-DNA
oligomers are extraordinarily stable A-T base-pairing systems
with a weak preference for the Hoogsteen pairing mode.17,18
Investigations on the enzymatic stability of tricyclo-DNA proved
their resistance against the 3′-exonuclease snake venom phos-
phodiesterase. Here we report on the synthesis of the hitherto
unknown tricyclodeoxycytidine and the tricyclodeoxyguanosine
building blocks 5 and 13, as well as their incorporation into
oligodeoxynucleotides. Moreover, X-ray structures of a R- and
a â-tricyclodeoxynucleoside, the pairing properties of fully
modified tricyclooligonucleotides containing all four bases, a
thermodynamic analysis of duplex formation, a CD spectro-
scopic analysis, and a modeling study of a tricyclo-DNA/DNA
duplex are presented.
a Reagents: (a) N 4-benzoylcytosine, CF3SO3SiMe3, BSA, CH3CN, 0 °C,
overnight, 93%; (b) TBAF, THF, 3 min, 84% (both isomers); (c) HF Et3N,
THF, 9 h, 90%; (d) DMTOTf, pyridine, 12 h, 87%; (e) 2-cyanoethyl N,N-
diisopropylchlorophosphoramidite, N,N-diisopropylethylamine, CH3CN, 2
h, 91%.
Lewis acid and BSA as the silylating agent. To circumvent the
formation of N 7/N 9-mixtures during nucleosidation,20 we first
chose the protected O6-p-nitrophenethyl-N 2-isobutyrylguanine
as the base synthon.21 Unfortunately, in this way only the
corresponding R-nucleosides in a N 7/N 9 ratio of 2:1 could be
obtained in 65% yield (see Supporting Information).
Results and Discussion
Synthesis of Tricyclodeoxycytidine and Tricyclodeoxygua-
nosine and Their Phosphoramidite Building Blocks. For
nucleoside synthesis the tricyclic sugar derivative 1 (Scheme
1), which had already been prepared in our laboratory, was
chosen as starting material.19 The Me3SiOTf-induced nucleosi-
dation of 1 with N 4-benzoylcytosine in CH3CN at 0 °C in the
presence of N,O-bis(trimethylsilyl)acetamide (BSA) afforded 2
in 93% yield as an anomeric mixture of R:â ) 1:1. Variation
of the reaction temperature did not affect the ratio of anomers.
Both anomers could be separated by chromatography after
selective removal of the Me3Si group at C(3′) with TBAF.
Unambiguous assignment of the R- and â-configuration in 3
and 4 was possible via 1H NMR difference NOE spectroscopy.
In addition, 3 could be crystallized and its structure confirmed
by X-ray analysis (vide infra). Treatment of 4 with HF/Et3N
afforded 5 in 90% yield. Selective tritylation of the tertiary OH
group at C(5′) was achieved with 4,4′-dimethoxytrityl trifluo-
romethane sulfonate (DMTOTf)14 in pyridine in 87% yield. The
phosphoramidite building block 7 was obtained by standard
reaction of 6 with 2-cyanoethyl N,N-diisopropyl-chlorophos-
phoramidite as a 3:2 diastereoisomeric mixture (1H and 31P
NMR).
Alternatively, the nucleosidation was realized with O6-
diphenyl-carbamoyl-N2-isobutyrylguanine as the protected base.22
Under these conditions, a rewarding N 7/N 9 ratio of 1:9 was
obtained from which the two anomers of the N 9-nucleosides, 8
and 9, were isolated in ca. 24% yield, each (Scheme 2).
Elimination of the O6-diphenyl-carbamoyl protecting group was
achieved by heating 9 in a saturated solution of NaNO2 in
DMSO to give 11 in 90% yield (Scheme 3).23 The â-configu-
1
ration in 11 could be assigned unambiguously by H NMR
difference NOE spectroscopy, while the N-9 connectivity was
warranted by the characteristic chemical shifts of C(4) and C(5)
in the 13C NMR spectra.20 After deprotection of the O(5′)-
TBDMS group using HF/Et3N in THF, the nucleoside analogue
12 became available. The phosphoramidite building block 14
was obtained by selective tritylation of 12 with DMTOTf in
pyridine followed by reaction with 2-cyanoethyl N,N-diisopro-
pylchlorophosphoramidite. 1H and 31P NMR indicated a mixture
of two diastereoisomers (at phosphorus) in a ratio of 3:2.
Structure of Tricyclodeoxynucleosides. To investigate
constitution and conformation of the tricyclodeoxynucleosides,
we subjected crystals of the R-tricyclodeoxycytidine derivative
3 as well as of N6Bz-â-tricyclodeoxyadenosine 15 (obtained as
described before19) to X-ray structure analysis. In both cases,
the expected exo-configuration of the cyclopropane ring and
the respective configuration at the anomeric center could be
The nucleosidation of the tricyclic sugar 1 with protected
guanine was attempted in the same way with Me3SiOTf as
(15) Bolli, M.; Litten, C.; Schu¨tz, R.; Leumann, C. Chem. Biol. 1996, 3, 197-
206.
(16) Bolli, M.; Trafelet, H. U.; Leumann, C. Nucleic Acids Res. 1996, 24, 4660-
(20) Garner, P. J. Org. Chem. 1988, 53, 1294-1298.
(21) Jenny, T. Nucleosides Nucleotides 1992, 11, 1257.
(22) Timar, Z.; Kova´cs, L.; Kova´cs, G.; Schmel, Z. J. Chem. Soc., Perkin Trans.
1 2000, 19-26.
4667.
(17) Steffens, R.; Leumann, C. J. J. Am. Chem. Soc. 1999, 121, 3249-3255.
(18) Steffens, R.; Leumann, C. J. J. Am. Chem. Soc. 1997, 119, 11548-11549.
(19) Steffens, R.; Leumann, C. HelV. Chim. Acta 1997, 80, 2426-2439.
(23) Ackermann, D.; Pitsch, S. HelV. Chim. Acta 2002, 85, in press.
9
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Watson−Crick Base Pairing of Tricyclo-DNA
A R T I C L E S
Scheme 2a
a Reagents: (a) O6-diphenyl-carbamoyl-N 2isobutyrylguanine, CF3SO3-
SiMe3, BSA, CH3CN, 50 °C, 3 h, 53%.
Scheme 3a
Figure 2. X-ray crystal structures of tricyclodeoxynucleosides: (a) 3 and
(b) 15.
Table 1. Furanose Conformation and Values of Selected Torsion
Angles of Tricyclonucleosides 3 and 15, Bicyclonucleoside â-bcd
T,26 and B-DNA27
3
15
â-bcd T
B-DNA
furanose pucker C(3′)-endo C(2′)-endo C(1′)-exo C(2′)-endo
γ
δ
ø
158°
96°
170°
130°
154°
59°
149°
126°
-113°
54°
123°
-117°
a Reagents: (a) sat. NaNO2 in DMSO, 60 °C, 10 h, 90%; (b) HF Et3N,
THF, 8 h, 61%; (c) DMTOTf, pyridine, 10 h, 83%; (d) 2-cyanoethyl N,N-
diisopropylchlorophosphoramidite, N,N-diisopropylethylamine, CH3CN, 10
h, 58%.
correction of γ, relative to bicyclonucleosides, is achieved by
introduction of the cyclopropane ring.
confirmed (Figure 2). In the R-nucleoside 3, the furanose ring
occurs in a C(3′)-endo conformation (3E, North-type). Although
there exists a trend for the preference of the C(3′)-exo, the C(4′)-
endo, and the C(2′)-exo sugar pucker in R-nucleosides, the C(3′)-
endo conformation, as found here, is not unique.24,25 Values
for the backbone relevant torsion angles γ, δ, and ø are
summarized in Table 1. Intermolecular contacts in the crystal
of 3 arise from H-bonds between N(4)-H‚‚‚O(3′) and O(3′)-
H‚‚‚N(3).
In the case of the â-nucleoside 15, the adenine base is syn-
oriented (ø ) 59°) and kept in this conformation via a intra-
molecular H-bond between O(5)-H and N(3). This intramo-
lecular H-bond almost certainly influences the conformation of
the furanose ring (C(2′)-endo, 2E, South-type) and torsion angle
γ (Table 1), rendering an extrapolation of the conformation of
â-tricyclodeoxynucleoside units within an oligonucleotide chain
difficult. It becomes clear, however, that only a marginal
Synthesis of the Oligonucleotides 16-25. Figure 3 gives
an overview on the oligonucleotides prepared for the present
study. Tricyclodeoxynucleotide phosphoramidites were coupled
using standard procedures. The important changes to the typical
synthesis protocols were the use of longer coupling times (6-
10 min) and the use of 5-(benzylmercapto)-1H-tetrazole instead
of tetrazole as the activator.
Detritylation was affected as usual (2% trichloroactetic acid,
60 s). Coupling yields were generally >98% according to trityl
assay. Oligomers 20 and 21 had to be 5′-phosphorylated, due
to the chemical instability of 5′-terminal, unprotected tricyclo-
deoxynucleoside residues under the conditions of deprotection
and cleavage from solid support.17 For reasons of comparability,
the oligomers 16-19 and 22, 23 were also 5′-phosphorylated.
Crude oligonucleotides were purified by DEAE ion-exchange
HPLC. Their purity was controlled by RP-HPLC before
analyzing their structural integrity by ESI-MS spectroscopy
(Figure 3).
(24) Sundaralingam, M. J. Am. Chem. Soc. 1971, 93, 6644-6647.
(25) Swarna, L. Y.; Yathindra, N. Biopolymers 1992, 32, 249-266.
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A R T I C L E S
Renneberg and Leumann
Table 2. Tm and ∆Tm Values (°C) from UV Melting Curves (260 nm)a
DNA complement
RNA complement
(150 mM NaCl)
(1 M NaCl)
(150 mM NaCl)
∆Tm
(1 M NaCl)
b
b
c
c
Tm
∆Tm
Tm
∆Tm
Tm
Tm
∆Tm
23: d(pAACTGTCACG)
16: d(pAACTGT c ACG)
17: d(pAACTG t CACG)
18: d(pAACTGTC a CG)
19: d(pAACT g TCACG)
44.4
46.8
45.3
43.4
44.7
49.4
52.5
51.4
49.1
50.4
42.5
45.7
43.0
42.1
43.5
47.8
49.8
48.2
47.0
47.7
+2.4
+0.9
-1.0
+0.3
+3.1
+2.0
-0.3
+1.0
+3.2
+0.5
-0.4
+1.0
+2.0
+0.4
-0.8
-0.1
a 10 mM NaH2PO4, pH 7.0, c ) 2 µM. b Values relative to DNA/DNA duplex. c Values relative to DNA/RNA duplex.
Table 3. Tm and ∆Tm Values (°C) from UV Melting Curves (260 nm) in a 150 mM NaCl (1 M NaCl in parentheses)a
22: d(pCGTGACAGTT)
20: tcd(pcgtgacagtt)
24: r (CGUGACAGUU)
Tm ∆Tm/mod.
Tm
∆Tm/mod.
Tm
∆Tm/mod.
23: d(pAACTGTCACG)
21: tcd(paactgtcacg)
25: r(AACUGUCACG)
44.4 (49.4)
55.2 (62.7)
40.1 (46.0)
57.2 (62.8)
75.2 (∼80)
64.8 (69.5)
+1.2 (+1.3)
+3.1 (+3)
+2.4 (+2.4)
42.5 (47.8)
61.6 (67.6)
52.2 (57.5)
+1.1 (+1.3)
+1.9 (+2.0)
a 10 mM NaH2PO4, pH 7, c ) 2 µM.
Figure 3. Sequences of tricyclodeoxynucleoside containing oligonucleotides
and their corresponding mass-spectrometric data (a ) tricyclodeoxy-
adenosine; c ) tricyclodeoxycytosine; g ) tricyclodeoxyguanosine; t )
tricyclothymidine).
Figure 4. UV melting curves (260 nm) of DNA/DNA, DNA/tricyclo-DNA,
and tricyclo-DNA/tricyclo-DNA duplexes: (a) 22/23, (b) 20/23, (c) 21/22,
(d) 20/21 in 10 mM NaH2PO4, pH 7, 150 mM NaCl, c ) 2 µM.
Duplex Formation of Oligonucleotides Containing Single
Tricyclodeoxynucleotide Units with DNA and RNA. Comple-
mentary pairing experiments of the sequences 16-19 were
carried out with DNA as well as RNA. The duplex formation
was monitored by UV melting curve analysis (260 nm). The
melting temperatures (Tm) are summarized in Table 2.
invokes the intrinsically weaker intrastrand stacking of pyrimi-
dine bases that is compensated more efficiently by the rigid
backbone in tricyclo-DNA, relative to DNA.
Duplex Formation of Fully Modified Oligotricyclodeoxy-
nucleotides with DNA and RNA. To determine the ability of
tricyclo-DNA to form stable Watson-Crick duplexes, fully
modified tricyclo-DNA oligonucleotides containing all four
bases in a random distribution were examined. Complementary
pairing experiments of tricyclo-DNA oligonucleotides 20, 21
were carried out in their own series and with natural DNA and
RNA at low (150 mM) and high (1 M) NaCl concentration. To
exclude self-pairing of 20 or 21, a UV melting experiment of
oligonucleotide 20 without complement was performed. As
expected, no cooperative melting transition at the temperature
interval of 0-90 °C could be observed, indicating the absence
of self-association.
From these data, it appears that single substitutions of the
four different tricyclodeoxynucleotide units within the oligomer
showed subtle but distinct differences in Tm relative to the
natural oligodeoxynucleotide. The oligonucleotide 16, containing
a tricyclodeoxycytidine unit, shows the highest increase in Tm.
At 150 mM NaCl, a ∆Tm of +2.4 °C with DNA and of +3.2
°C with RNA as the complement was measured. Duplexes
containing a tricyclodeoxythymine or tricyclodeoxyguanosine
unit still showed increased Tm (+0.3 to +1.0 °C), while
incorporation of a tricyclodeoxyadenosine unit leads to a duplex
with slightly decreased stability (-0.4 to -1.0 °C) relative to
the unmodified duplex. The observed differences in stability
might be either due to differential structural effects intrinsic to
the tricyclo-modification or due to differential nearest neighbor
effects in the sequence. As a general picture, it emerges that
tricyclopyrimidine nucleotides stabilize duplex formation with
complementary DNA and RNA more than tricyclopurine
nucleosides. A possible rationalization of this observation
The melting temperatures and the corresponding melting
curves for duplexes with complementary DNA are depicted in
Table 3 and Figure 4. Both duplexes containing one tricyclo-
DNA strand, 20/23 and 21/22, show increased melting temper-
atures with a ∆Tm of +11-12 °C as compared to the natural
systems. Thus, the stabilization per modification (∆Tm/mod.)
amounts to +1.2 °C. In its own series, tricyclo-DNA constitutes
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5996 J. AM. CHEM. SOC. VOL. 124, NO. 21, 2002

Watson−Crick Base Pairing of Tricyclo-DNA
A R T I C L E S
Table 4. Tm and ∆Tm Values of Mismatched versus Matched Duplexes within the Backbone Series Tricyclo-DNA, DNA, and RNA, against
RNA as a Target (10 mM NaH2PO4, 150 mM NaCl, pH 7.0, c ) 2 µM)
r(UUGACXGUGC)
X ) A
X) U
X) G
X) C
Tm
Tm
∆Tm
Tm
∆Tm
Tm
∆Tm
24 (RNA)
22 (DNA)
20 (tc-DNA)
52.2 °C
42.5 °C
61.6 °C
39.5 °C
27.5 °C
44.1 °C
-12.7 °C
-15.0 °C
-17.5 °C
50.6 °C
40.4 °C
59.0 °C
-1.6 °C
-2.1 °C
-2.6 °C
39.2 °C
28.0 °C
46.0 °C
-13.0 °C
-14.5 °C
-15.6 °C
Analysis of the Helix Conformation of Tricyclooligonu-
cleotide Duplexes. A CD spectroscopic analysis of tricyclo-
deoxyoligonucleotide-containing duplexes was performed to
investigate the structural preferences. CD spectra above and
below the melting temperature were recorded for the pure
tricyclo-DNA duplex 20/21, the tricyclo-DNA/DNA hybrid
duplexes 21/22 and 20/23, the tricyclo-DNA/RNA hybrid 21/
24, as well as for the control DNA duplex 22/23, and the DNA/
RNA duplex 23/24 (Figure 6).
In the nondenatured state, the CD spectra of the pure tricyclo-
DNA or the DNA/tricyclo-DNA duplexes (B, C, D) show
hypsochromic shifts of the ellipticity maximum by about 9-17
nm, relative to the DNA duplex (A). In all these cases, only a
minor change in intensity of the negative minimum ellipticity
at ca. 240 nm is observed upon denaturation, which is in contrast
to the spectra of the pure DNA duplex. The proportion of the
intensity of the positive over the negative maximum is in the
range from 2:1 to 4:1 in the tricyclo-DNA-containing duplexes,
while it is slightly less than 1:1 for the DNA duplex. The CD
spectra of the pure tricyclo-DNA duplex (B) and the tricyclo-
DNA hybrid duplexes (C, D) are thus reminiscent of a helix of
the A-conformation, while that of the DNA duplex (A) showed,
as expected, all of the characteristics of a B-helix. The CD
spectra of the corresponding RNA/tricyclo-DNA duplex (F) are
very similar to that of the RNA/DNA reference spectra,
indicating again A-conformation of the helices. The structural
preference of tricyclo-DNA for A-type double helices is in line
with the enhanced stability of tricyclo-DNA/RNA duplexes
relative to tricyclo-DNA/DNA duplexes.
Figure 5. UV melting profiles (260 nm) of DNA/RNA and tricyclo-DNA/
RNA duplexes: (a) 22/25, (b) 23/24, (c), 20/25, (d) 21/24 in 10 mM NaH2-
PO4, pH 7, 150 mM NaCl, c ) 2 µM single strand concentration.
the most stable pairing systems with a Tm of 75 °C for the
decamer duplex 20/21 (∆Tm of +31 °C). As expected, higher
salt concentration leads to higher melting temperatures for all
duplexes. No differential effects between DNA and the tricyclo-
DNA were observed, indicating a similar dependence of Tm on
electrolyte concentration.
With complementary RNA, duplex stabilization was even
more pronounced. As extracted from Table 3 and Figure 5, both
tricyclo-DNA/RNA heteroduplexes show a considerably higher
Tm as compared to the natural hybrid. For the duplex 20/25, an
increase in stability of more than 24 °C was observed which
results in a ∆Tm/mod. ) +2.4 °C. Remarkably, the heterodu-
plexes formed between tricyclo-DNA and RNA are more stable
than the corresponding natural RNA duplex by ca. 10-12 °C.
Mismatch Discrimination in Tricyclo-DNA/DNA and
Tricyclo-DNA/RNA Duplexes. To analyze the base selectivity
in RNA recognition by tricyclo-DNA, we investigated the
duplexes between complementary RNA, containing one mis-
matched base in the center of the sequence, with 20 (tricyclo-
DNA) and compared it to those with 22 (DNA) and 24 (RNA).
As can be seen from the ∆Tm values in Table 4, any mismatched
base arrangement in the tricyclo-DNA/RNA hybrid duplexes
is associated with a dramatic decrease in Tm giving rise to ∆Tm
values of -17.5 °C for the T-U and -15.6 °C for the T-C
arrangement relative to the standard T-A base pair. As
expected, the T-G wobble base pair is only slightly less stable
than the matched T-A base pair in all cases investigated.
Considering the various backbones involved, relative mismatch
discrimination is largest in the tricyclo-DNA/RNA hybrids
intermediate in the DNA/RNA hybrids and lowest in the RNA
duplexes. From these experiments, it appears that tricyclo-DNA
does not only recognize complementary RNA with increased
thermal stability, but also with increased selectivity.
Molecular Dynamics Simulation. The preference of tricyclo-
DNA/DNA duplexes for the A-conformation, as determined by
CD spectroscopy, prompted us to perform a molecular dynamics
simulation of the duplex 21/22 to get independent support and
to determine the backbone parameters. The simulation started
from a duplex in B-DNA geometry and resulted after a 200 ps
trajectory in a double helical structure which strongly resembles
that of an A-form helix. An average structure of the last 50 ps
of the internal six base pairs of the decamer is depicted in Figure
7. For comparison, the corresponding bicyclo-DNA/DNA duplex
of the same sequence was analyzed in analogy. The structural
evaluation of the two systems shows an interesting picture
concerning the six torsion angles describing the repeating unit
of the phosphodiester backbone (Table 5) and highlights the
structural effect of the cyclopropane ring on the bicyclic core
system.
The phosphodiester bonds in the tricyclo-DNA strand (torsion
angle R and ú) are in a trans/gauche arrangement and thus differ
from those of the other duplexes. A remarkable change was
found for the torsion angle â which shows values of 87° in
tricyclo-DNA. This is in contrast to the ca. 180° typically
observed in A- or B-DNA. The furanose ring prefers a O(4′)-
9
J. AM. CHEM. SOC. VOL. 124, NO. 21, 2002 5997

A R T I C L E S
Renneberg and Leumann
Figure 6. CD traces of duplexes containing DNA and tricyclo-DNA strands at different temperatures: (A) 22/23; (B) 20/21; (C) 20/23; (D) 21/22; (E)
23/24; (F) 21/24 in 10 mM NaH2PO4, 150 mM NaCl, pH 7, c ) 2 µM.
Table 5. Backbone Torsion Angles R-ú for A- and B-DNA as well
as for Bicyclo-DNA and Tricyclo-DNA in Complex with Natural
DNA, Determined by Molecular Modeling
a
a
torsion angles
A-DNA
B-DNA
bicyclo-DNA
tricyclo-DNA
R
â
γ
δ
ꢀ
-75° ( 36
-175° ( 13
56° ( 22
-63° ( 8
171° ( 14
54° ( 8
-166° ( 2 -156° ( 10
178° ( 2
143° ( 4
122° ( 3
177° ( 3
-86° ( 4
87° ( 6
149° ( 10
92° ( 7
173° ( 8
23° ( 6
91° ( 18
123° ( 21
-166° ( 19 -169° ( 25
-75° ( 19 -108° ( 34
ú
Figure 7. Stereoscopic view of the averaged structure of the last 50 ps of
the 200 ps molecular dynamics simulation (Insight II/Discover) of the six
central base pairs of the duplex 21/24.
a Data taken from ref 27.
structural bias originating from the syn orientation of the adenine
base enforced via intramolecular H-bonding (vide supra) in the
crystal of 15.
From comparison of the data of bicyclo- and tricyclo-DNA,
it appears that the cyclopropane ring has an astonishingly minor
effect on torsion angle γ. However, it drives the furanose ring
of the bicyclic core system preferentially into a C(3′)-endo-
endo conformation. The torsion angle γ ) 149° is in the anti
orientation, while δ ) 92° is in a gauche arrangement, as
observed for A-DNA. The structural differences of the modeled
tricyclo-DNA units in the furanose pucker and torsion angles γ
and δ with those discernible from the X-ray crystal structure of
the tricyclodeoxynucleoside 15 are best explained by the
9
5998 J. AM. CHEM. SOC. VOL. 124, NO. 21, 2002

Watson−Crick Base Pairing of Tricyclo-DNA
A R T I C L E S
Table 6. Thermodynamic Data of Duplex Formation Determined
from the 1/Tm versus ln(c) Plots in a 10 mM NaH2PO4, 150 mM
NaCl, pH 7 Buffer
25 °C
∆H
∆S
∆G
(kcal/mol)
(cal/mol K)
(kcal/mol)
DNA 22/DNA 23
-68.2
-63.8
-65.4
-117.0
-187.8
-167.2
-170.8
-308.1
-12.2
-14.0
-14.5
-25.2
DNA 23/tc-DNA 20
tc-DNA 21/DNA 22
tc-DNA 20/tc-DNA 21
less strain in the duplex with the homostructural tricyclo-
backbone, one cannot exclude at this point whether the data
are biased by non-two-state model behavior of tricyclo-DNA
duplex melting. For clarification, model independent thermo-
dynamic data, as, for example, from calorimetric experiments,
are necessary.
Figure 8. Nucleoside units and idealized Newman projections along the
O(5′)-C(5′) bond (torsion angle â) of the averaged structures as obtained
from molecular modeling: (A) B-DNA; (B) bicyclo-DNA; (C) tricyclo-
DNA.
Conclusions
Fully modified tricyclo-DNA sequences form remarkably
stable duplexes with DNA as well as with RNA. The average
increase in melting temperature was determined to be +1.2 °C/
mod. with complementary DNA and +2.4 °C/mod. with
complementary RNA. The most stable duplexes, however, are
formed in the pure tricyclo-DNA series with ∆Tm values of +3.1
°C/base pair relative to DNA. Base-base recognition is highly
selective in tricyclo-DNA/RNA duplexes and is superior to that
of pure RNA and DNA/RNA duplexes. In the homoadenine-
homothymine sequence context, tricyclo-DNA can show in-
creases in Tm of up to +4 °C/mod. In the latter cases, however,
the Hoogsteen pairing modus is preferred over the Watson-
Crick base-pairing modus. In comparison to other conforma-
tionally restriced oligonucleotide analogues, tricyclo-DNA
shows higher RNA affinity as the recently introduced CeNA29
and is only slightly superseded by the analogue HNA,12,13 which
displays increases in Tm of +1.3 °C/mod. with complementary
DNA and +3 °C/mod. with RNA. With respect to LNA,9,11
about one-half of the increase in thermal stability (∆Tm/mod.
) 4-8 °C against RNA) can be gained. Tricyclo-DNA also
shows enhanced binding efficiency relative to DNA analogues
with charge neutral backbones, as PNA30 or the morpholino-
DNA.31
In comparison to bicyclo-DNA, a remarkably higher propen-
sity for Watson-Crick duplex formation is found for tricyclo-
DNA. A molecular modeling study and CD spectroscopic
analysis of a tricyclo-DNA/DNA duplex revealed a strong
preference for an A-type helix, assigning tricyclo-DNA, in
contrast to bicyclo-DNA, the character of an RNA mimic. In
the absence of high-resolution structural data, it appears from
molecular modeling and CD spectroscopy that the cyclopropane
ring drives the furanose unit into a N-type conformation and
torsion angle â into a gauche instead of trans arrangement. These
changes seem to be primarily responsible for the overall A-helix
shape of duplexes and for the enhanced stability of Watson-
Crick duplexes relative to bicyclo-DNA. Alternatively, the
higher hydrophobicity of the tricyclo-DNA system along with
Figure 9. 1/Tm vs ln(c) plots of the four duplex systems (10 mM NaH2-
PO4, 150 mM NaCl, pH 7.0).
like conformation and is responsible for the observed change
in torsion angle â. A conformational analysis around the O(5′)-
C(5′) bond illustrates that in tricyclo-DNA the gauche arrange-
ment of â is preferred over the typical trans arrangement, due
to the steric constraints imposed by the cyclopropane ring
(Figure 8). We assume that the preferential change of â from
trans to gauche going from bicyclo- to tricyclo-DNA is
responsible for the improved Watson-Crick base-pairing prop-
erties of the latter system. Although four out of six backbone
torsion angles differ appreciably from that of A-DNA (or RNA),
the overall shape of the double helix still preserves an A-like
character, a fact that is fully supported by the CD spectroscopic
investigations. Thus, tricyclo-DNA, in contrast to bicyclo-DNA,
is a structural mimic of RNA and not of DNA.
Thermodynamic Data of Duplex Formation. Thermody-
namic data of duplex formation were obtained from DNA
melting curves by the concentration variation method.28 The
data obtained from the 1/Tm versus ln(c) plots of the four duplex
systems (Figure 9) are summarized in Table 6. The calculated
standard enthalpies of duplex formation ∆G25 °C are in complete
agreement with the observed thermal stabilities of the four
systems. The two tricyclo-DNA/DNA duplexes show an entropic
stabilization along with an enthalpic destabilization, relative to
the natural system. However, the pure tricyclic duplex behaves
differently. Here the stability appears to be largely enthalpy
driven. Although it is tempting to interpret the data in terms of
(29) (a) Wang, J.; Verbeure, B.; Luyten, I.; Lescrinier, E.; Froeyen, M.; Hendrix,
C.; Rosemeyer, H.; Seela, F.; Van Aerschot, A.; Herdewijn, P. J. Am. Chem.
Soc. 2000, 122, 8595-8602. (b) Verbeure, B.; Lescrinier, E.; Wang, J.;
Herdewijn, P. Nucleic Acids Res. 2001, 29, 4941-4947.
(26) Tarko¨y, M.; Bolli, M.; Schweizer, B.; Leumann, C. HelV. Chim. Acta 1993,
76, 481-510.
(27) Saenger, W. Principles of Nucleic Acid Structure; Springer-Verlag: New
York, 1984.
(30) Uhlmann, E.; Peyman, A.; Breipohl, G.; Will, D. W. Angew. Chem., Int.
Ed. 1998, 37, 2796-2823.
(28) Marky, L. A.; Breslauer, K. J. Biopolymers 1987, 26, 1601-1620.
(31) Summerton, J. E. Biochim. Biophys. Acta 1999, 1489, 141-158.
9
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A R T I C L E S
Renneberg and Leumann
J ) 7.7 Hz), 7.61, 7.72 (2m, 3H), 8.06 (d, 2H, J ) 7.0 Hz), 8.43 (d,
1H, J ) 7.7 Hz). H NMR-difference-NOE: 4.45 (s, 1H) f 8.43 (d,
a differential hydration behavior of the helix, relative to bicyclo-
DNA, could also be in part responsible for the observed
differences in the hybridization properties.
In conclusion, with its favorable DNA and RNA binding
properties, tricyclo-DNA definitely represents an interesting
candidate worthy of further exploration. The investigation of
its antisense properties in vitro and in vivo will be the subject
of a following communication.
1
1H, J ) 7.7 Hz); 6.22 (t, 1H, J ) 6.3 Hz) f 2.27 (dd, 1H, J1 ) 6.3,
J2 ) 13.6 Hz), 2.84 (dd, 1H, J1 ) 6.3, J2 ) 13.6 Hz); 8.43 (d, 1H,
J ) 7.7 Hz) f 4.45 (s, 1H), 6.22 (t, 1H, J ) 6.3 Hz), 7.27 (d, 1H,
J ) 7.7 Hz). 13C NMR (75 MHz, CD3OD): δ -3.22, -3.07, 19.08,
19.33, 25.85, 26.56, 41.20, 48.65, 66.95, 87.28, 89.72, 91.93, 99.04,
129.62, 130.24, 134.69, 134.83, 146.44, 158.21, 164.68, 169.64. MS
(ESI-TOF): m/z (rel intensity) 482.20, 148.95. Data of 4: Rf 0.15
1
(EtOAc). H NMR (300 MHz, CD3OD): δ 0.22, 0.30 (2s, 6H), 0.92
Experimental Section
(m, 1H), 0.99 (s, 9H), 1.15 (m, 1H), 1.46 (m, 1H), 1.72 (d, 1H, J )
14.3 Hz), 1.84 (dd, 1H, J1 ) 4.4, J2 ) 14.3 Hz), 2.69 (m, 2H), 4.38 (s,
1H), 6.13 (d, 1H, J ) 6.3 Hz), 7.57 (m, 3H), 7.60 (m, 1H), 8.04 (d,
2H, J ) 7.0 Hz), 8.87 (d, 1H, J ) 7.4 Hz). 1H NMR-difference-NOE:
8.87 (d, 1H, J ) 7.4 Hz) f 1.46 (m, 1H), 6.13 (d, 1H, J ) 6.3 Hz),
7.57 (m, 3H). 13C NMR (300 MHz, CD3OD): δ -3.21, -3.15, 18.11,
18.99, 26.01, 26.49, 42.91, 49.12, 67.12, 87.10, 92.11, 94.14, 97.73,
129.46, 130.11, 134.38, 134.98, 146.51, 157.93, 165.25, 169.43. IR
(film): 3397, 1652, 1482, 1304 cm-1. MS (LSIMS): m/z (rel intensity)
484 (17), 330 (20), 225 (29), 216 (100). HRMS (LSIMS): m/z calcd
for C25H34N3O5Si, 484.2267; found, 484.2266.
General. 1H NMR (300, 400, or 500 MHz) and 13C NMR (75, 100,
or 125 MHz) spectra were recorded on a Bruker AC-300, AMX-400,
or DRX500 spectrometer. 31P NMR spectra were measured on Bruker
DRX500 using 85% H3PO4 ()0 ppm) as external standard. Infrared
(IR) spectra were obtained using a Perkin-Elmer FTIR 1600 spectrom-
eter. Electron ionization (EI) mass spectral analyses were recorded on
a Varian MAT CH-7A; LSIMS on Micromass Autospec Q, matrix:
3-nitrobenzyl alcohol; ESI-MS on VG platform Fisons Instruments,
Single Stage Quadrupole ES-MS; and ESI-TOF-MS on Applied
Biosystems/Sciex QSTAR Pulsar (QqTOF) mass spectrometer. Flash
chromatography was performed using silica gel with an average particle
size of 40 µm. Chemicals, solvents, and reagents for reactions were
generally obtained from Fluka AG (Buchs, Switzerland), and were of
the highest quality available. All reactions were carried out under Ar.
(5′R)-(N 4-Benzoyl-1-[3′-O-trimethylsilyl-5′-O-(tert-butyldimeth-
ylsilyl)-2′-deoxy-3′-C, 5′-C-[(5′-C, 6′-C-methano)ethano]-D-ribofura-
nosyl]cytosine (2). To a suspension of N 4-benzoylcytosine (418 mg,
1.94 mmol) in CH3CN (9 mL) was added BSA (N,O-bis(trimethylsilyl)-
acetamide; 0.79 mL, 3.22 mmol). After stirring for 1 h at room
temperature, a solution of 1 (194 mg, 0.64 mmol) in CH3CN (5 mL) at
0 °C was added followed by CF3SO3SiMe3 (0.35 mL, 1.94 mmol).
After stirring for 4 h at 0 °C, another portion of CF3SO3SiMe3 (0.05
mL, 0.32 mmol) was added, and the reaction mixture was left overnight
at 4 °C. The solution was diluted with EtOAc and extracted with
saturated NaHCO3. The organic layer was dried over MgSO4 and
concentrated. Flash chromatography (hexane/EtOAc 1:4) of the residue
afforded 313 mg (87%) of 2 as a white foam in an anomeric mixture
R:â 1:1, besides 12 mg (4%) of 3 and 5 mg (2%) of 4 as colorless oils.
Data of 2: Rf 0.44 (hexane/EtOAc 1:4). 1H NMR (300 MHz, CDCl3):
δ 0.04 (s, 6H), 0.06 (s, 6H), 0.12 (s, 9H), 0.14 (s, 9H), 0.80 (m, 1H),
0.86 (s, 9H), 0.90 (s, 9H), 1.00 (m, 3H), 1.36, 1.54 (2m, 2H), 1.70 (m,
3H), 2.07 (dd, 1H, J1 ) 6.0, J2 ) 13.6 Hz), 2.28 (dd, 1H, J1 ) 5.2,
J2 ) 13.9 Hz), 2.70 (m, 2H), 2.95 (dd, 1H), 4.28 (s, 1H), 4.32 (s, 1H),
6.09 (m, 2H), 7.50 (m, 8H), 7.89 (d, 4H, J ) 7.0 Hz), 8.07 (d, 1H,
J ) 7.4 Hz), 8.70 (d, 1H), 8.87 (br, 2H). 13C NMR (75 MHz, CDCl3):
δ -3.80, -3.77, -3.71, 1.81, 1.85, 16.75, 18.69, 17.80, 23.91, 24.43,
25.65, 39.49, 42.09, 47.23, 47.41, 64.75, 65.15, 87.77, 87.88, 88.23,
90.63, 91.66, 93.94, 95.41, 96.21, 127.45, 127.50, 128.94, 128.95,
133.04, 143.64, 145.34, 162.20, 162.38. IR (film): 3007, 1652, 1478
cm-1. MS (EI): m/z (rel intensity) 556 (15), 341 (30), 297 (30), 216
(100).
(5′R)-(N 4-Benzoyl-1-[2′-deoxy-3′-C, 5′-C-[(5′-C, 6′-C-methano)-
ethano]-â-D-ribo-furanosyl]cytosine (5). To a solution of 4 (115 mg,
0.24 mmol) in THF (3 mL) was added HF Et3N (0.16 mL, 0.36 mmol).
After 6 h and 7 h, further portions of 0.5 equiv of HF Et3N were added.
After 9 h, the reaction mixture was diluted with EtOAc, adsorbed on
silica gel (1 g), and the solvent evaporated. Purification by flash
chromatography (EtOAc/EtOH 9:1) afforded 78 mg (90%) of 5 as a
1
white foam. Rf 0.38 (EtOAc/EtOH 9:1). H NMR (300 MHz, CD3-
OD): δ 0.93 (m, 1H), 1.04 (m, 1H), 1.56 (m, 1H), 1.69 (d, 1H, J )
14.3 Hz), 1.90 (dd, 1H, J1 ) 4.8, J2 ) 14.3 Hz), 2.47 (dd, 1H, J1 )
3.0, J2 ) 14.0 Hz), 2.73 (dd, 1H, J1 ) 7.0, J2 ) 14.0 Hz), 4.31 (s, 1H),
6.14 (dd, 1H, J1 ) 2.6, J2 ) 7.0 Hz), 7.54 (m, 2H), 7.64 (m, 2H), 7.97
(m, 2H), 8.65 (d, 1H, J ) 7.7 Hz). 13C NMR (75 MHz, CD3OD): δ
18.13, 25.33, 42.95, 49.00, 65.20, 86.54, 91.07, 93.10, 98.09, 129.41,
130.04, 134.86, 146.39, 157.83, 165.02, 169.22. IR (KBr): 3397, 1702,
1645, 1485, 1308, 1255 cm-1. MS (LSIMS): m/z (rel intensity) 370
(17), 341 (30), 216 (100), 155 (40), 119 (65). HRMS (LSIMS): m/z
calcd for C19H20N3O5, 370.1402; found, 370.1395.
(5′R)-(N 4-Benzoyl-1-[5′-O-(4,4′-dimethoxytriphenyl)methyl-2′-
deoxy-3′-C, 5′-C-[(5′-C, 6′-C-methano)ethano]-â-D-ribofuranosyl]-
cytosine (6). To a solution of 5 (175 mg, 0.47 mmol) in pyridine (1.9
mL) was added under stirring DMTOTf (435 mg, 0.95 mmol). After
4, 7, and 10 h, further portions of 1 equiv of DMTOTf were added.
The reaction mixture was diluted with EtOAc and extracted with
saturated NaHCO3. The organic layers were dried over MgSO4 and
concentrated. Purification by flash chromatography (EtOAc/EtOH
9:1 + 1% Et3N) afforded 277 mg (87%) of 6 as yellow foam. Rf 0.45
(EtOAc/EtOH 9:1). 1H NMR (300 MHz, CDCl3): δ 0.53 (m, 1H), 1.37
(m, 1H), 1.62 (d, 1H, J ) 13.6 Hz), 1.76 (m, 2H), 2.24 (s, 1H), 2.52
(d, 2H, J ) 4.0 Hz), 2.64 (s, 1H), 3.79, 3.80 (2s, 6H), 5.96 (t, 1H, J )
4.0 Hz), 6.83 (m, 4H), 7.25 (m, 3H), 7.40 (d, 4H), 7.51 (m, 4H), 7.61
(m, 1H), 7.93 (d, 2H), 8.79 (br, 1H), 9.00 (d, 1H, J ) 7.4 Hz). 13C
NMR (75 MHz, CDCl3): δ 14.16, 25.52, 41.49, 48.41, 54.82, 54.85,
68.06, 86.49, 88.50, 90.21, 92.09, 113.32, 127.15, 128.83, 128.97,
131.40, 132.68, 133.81, 137.45, 137.53, 144.91, 147.29, 159.34, 163.26.
IR (film): ν 3028, 1482 cm-1. MS (LSIMS): m/z (rel intensity) 672
(5′R)-(N 4-Benzoyl-1-[5′-O-(tert-butyldimethylsilyl)-2′-deoxy-3′-
C, 5′-C-[(5′-C, 6′-C-methano)ethano]-D-ribofuranosyl]cytosine (3 and
4). A solution of 2 (360 mg, 0.64 mmol) in THF (6 mL) and Bu4NF
(0.68 mL, 0.68 mmol, 1 M in THF) was stirred for 3 min. The reaction
was stopped by the addition of H2O, and the aqueous phase was
extracted with EtOAc. The combined organic phases were dried over
MgSO4 and concentrated. Flash chromatography (EtOAc) afforded 133
mg (43%) of 3 as a white solid and 128 mg (41%) of 4 as a white
foam. Crystals of 3 suitable for X-ray structure determination were
obtained from EtOAc. Data of 3: Rf 0.27 (EtOAc). 1H NMR (300 MHz,
CD3OD): δ 0.17, 0.21 (2s, 6H), 0.91 (s, 9H), 0.97 (m, 1H), 1.05 (m,
1H), 1.62 (m, 1H), 1.70 (d, 1H, J ) 13.6 Hz), 2.27 (dd, 1H, J1 ) 6.3,
J2 ) 13.6 Hz), 2.35 (dd, J1 ) 4.8, J2 ) 13.6 Hz), 2.84 (dd, 1H, J1 )
6.3, J2 ) 13.6 Hz), 4.45 (s, 1H), 6.22 (t, 1H, J ) 6.3 Hz), 7.27 (d, 1H,
(40), 530 (17), 455 (12). MS (ESI-TOF): m/z calcd for [M - H]-
)
670.2553; found, 670.2557.
(5′R)-(N 4-Benzoyl-1-[3′-O-(2-cyanoethoxy)(diisopropylamino)-
phosphino-5′-O-(4,4′-dimethoxytriphenyl)methyl-2′-deoxy-3′-C, 5′-
C-[(5′-C, 6′-C-methano)ethano]-â-D-ribofuranosyl]cytosine (7). 2-Cy-
anoethyl N,N-diisopropylchlorophosphoramidite (234 µL, 1.13 mmol)
was added dropwise at room temperature to a solution of 6 (253 mg,
0.37 mmol) and N,N-diisopropylethylamine (0.32 mL, 1.88 mmol) in
CH3CN (2.3 mL). After 2 h, EtOAc was added, and the mixture was
9
6000 J. AM. CHEM. SOC. VOL. 124, NO. 21, 2002

Watson−Crick Base Pairing of Tricyclo-DNA
A R T I C L E S
extracted with saturated NaHCO3. The combined organic layers were
dried over MgSO4 and concentrated. Flash chromatography (EtOAc +
1% Et3N) yielded 300 mg (91%) of 7 as a slightly yellow foam (1:1
1.27 (2s, 6H), 1.45 (m, 1H), 1.62 (d, 1H, J ) 14.0 Hz), 1.72 (dd, 1H,
J1 ) 4.8, J2 ) 14.3 Hz), 2.74 (m, 2H), 2.92 (dd, 1H, J1 ) 2.2, J2 )
13.6 Hz), 4.31 (s, 1H), 6.35 (dd, 1H, J1 ) 2.2, J2 ) 6.6 Hz), 8.50 (s,
1H). 1H NMR difference NOE: 1.45 (m, 1H) f 8.50 (s, 1H); 4.31 (s,
1H) f 0.83 (m, 1H), 2.74 (m, 2H), 6.35 (dd, 1H, J1 ) 2.2, J2 ) 6.6
Hz); 6.35 (dd, 1H, J1 ) 2.2, J2 ) 6.6 Hz) f 2.74 (m, 2H), 4.31 (s,
1H), 8.50 (s, 1H); 8.50 (s, 1H) f 1.45 (m, 1H), 6.35 (dd, 1H, J1 )
2.2, J2 ) 6.6 Hz). 13C NMR (75 MHz, CD3OD): δ -3.19, -3.13,
18.66, 19.03, 19.66, 19.67, 26.30, 37.27, 42.75, 49.10, 67.10, 88.01,
89.10, 93.61, 122.44, 139.60, 139.70, 149.79, 157.81, 182.04. MS (ESI-
TOF): m/z calcd for C23H34N5O5Si, 488.2329; found, 488.2326.
(5′R)-(N 2-Isobutyryl-9-[2′-deoxy-3′-C, 5′-C-[(5′-C, 6′-C-methano)-
ethano]-â-D-ribo-furanosyl]guanine (12). A solution of 11 (145 mg,
0.30 mmol) in THF (6 mL) and HF Et3N (0.3 mL, 0.59 mmol) was
stirred for 8 h. Silica gel (0.5 g) was added, and the solvent evaporated
to dryness. Flash chromatography (EtOAc/EtOH 4:1 f 3:2) yielded
68 mg (61%) of 12 as a yellow solid. Rf 0.51 (CH2Cl2/MeOH 5:1). 1H
NMR (300 MHz, CD3OD): δ 0.86 (m, 1H), 1.04 (m, 1H), 1.24, 1.27
(2s, 6H), 1.54 (m, 1H), 1.66 (d, 1H, J ) 14.0 Hz), 1.95 (dd, 1H, J1 )
5.1, J2 ) 14.0 Hz), 2.70 (m, 2H), 2.79 (dd, 1H, J1 ) 4.0, J2 ) 13.6
Hz), 4.24 (s, 1H), 6.36 (dd, 1H, J1 ) 4.0, J2 ) 6.6 Hz), 8.42 (br, 1H).
13C NMR (75 MHz, DMSO-d6): δ 17.29, 19.04, 19.39, 23.26, 34.86,
41.29, 47.12, 63.11, 85.33, 85.38, 90.80, 121.00, 137.43, 147.91, 147.99,
154.99, 180.24. IR (KBr): 3240, 2965, 1676, 1611 cm-1. MS
(LSIMS): m/z (rel intensity) 376 (4), 242 (100), 222 (12). HRMS
(LSIMS): m/z calcd for C17H22N5O5, 376.1620; found, 376.1613.
1
mixture of diastereoisomers). Rf 0.36, 0.47 (EtOAc). H NMR (400
MHz, C6D6): δ 0.70 (m, 0.6H), 0.75 (m, 0.4H), 0.95 (m, 1H), 1.10
(m, 12H), 1.33 (m, 2H), 1.57 (m, 1H), 1.83 (m, 2H), 2.06 (d, 1H, J )
14.3 Hz), 2.21 (dd, 1H, J1 ) 3.7, J2 ) 14.3 Hz), 2.68 (dd, 1H, J1 )
14.0, J2 ) 20.6 Hz), 2.89 (dd, 0.6H, J1 ) 7.0, J2 ) 14.3 Hz), 3.01 (m,
0.4H), 3.04 (s, 0.6H), 3.06 (s, 0.4H), 3.21 (m, 2H), 3.44, 3.45 (2s,
6H), 6.15 (d, 1H, J ) 5.1 Hz), 6.90 (m, 4H), 7.13 (m, 4H), 7.31 (m,
3H), 7.65 (m, 5H), 7.80 (d, 2H, J ) 7.4 Hz), 7.97 (br, 1H), 9.10 (m,
1H). 13C NMR (100 MHz, C6D6): δ 18.05, 20.05, 20.13, 24.40, 24.48,
24.56, 24.64, 24.71, 25.62, 39.65, 43.48, 43.54, 43.61, 43.67, 46.54,
54.99, 58.06, 58.26, 58.39, 58.59, 67.75, 88.87, 88.90, 90.37, 90.44,
90.49, 90.56, 92.38, 92.68, 113.54, 117.67, 117.85, 127.42, 127.42,
128.22, 128.49, 128.89, 131.64, 132.64, 137.56, 137.59, 144.50, 147.35,
147.38, 159.06, 163.42. 31P NMR (161.9 MHz, C6D6): δ 143.96,
145.18. IR (KBr): 1663, 1507, 1485, 1244 cm-1. MS (ESI-TOF): m/z
calcd for [M - H]- ) 870.3632; found, 870.3627.
(5′R)-(O6-Diphenylcarbamoyl-N 2-isobutyryl-[3′-O-trimethylsilyl-
5′-O-(tert-butyldimethylsilyl)-2′-deoxy-3′-C, 5′-C-[(5′-C, 6′-C-metha-
no)ethano]-D-ribofuranosyl]guanine (8-10). A suspension of O6-
diphenylcarbamoyl-N 2-isobutyrylguanine (1.82 g, 4.35 mmol) in
CH3CN (20 mL) and BSA (4.4 mL, 18.14 mmol) was stirred for 30
min at 40 °C. To the clear solution was added at room temperature 1
(1.09 g, 3.62 mmol) in CH3CN (10 mL) followed by CF3SO3SiMe3
(1.94 mL, 10.86 mmol). The reaction mixture was stirred for 3 h at 50
°C. The reaction was stopped by the addition of EtOAc and extraction
with saturated NaHCO3. The combined organic layers were dried over
MgSO4 and concentrated. Flash chromatography (hexane/EtOAc
2:1 f EtOAc f MeOH) afforded 528 mg (20%) of 8, 123 mg (5%)
of des-trimethylsilyl-8, 195 mg (7%) of 9, 370 mg (15%) of des-
trimethylsilyl-9, and 130 mg (5%) 10 (mixture of R- and â-anomers)
as white foams. Data of 8: Rf 0.49 (hexane/EtOAc 2:1). 1H NMR (300
MHz, DMSO-d6): δ 0.04 (s, 6H), 0.14 (s, 9H), 0.73 (m, 1H), 0.82 (s,
9H), 0.99 (m, 1H), 1.10, 1.12 (2s, 6H), 1.43 (m, 1H), 1.63 (d, 1H, J )
14.0 Hz), 2.04 (m, 1H), 2.66 (dd, 1H, J1 ) 7.4, J2 ) 13.6 Hz), 2.88
(m, 1H), 3.22 (dd, 1H, J1 ) 4.0, J2 ) 13.6 Hz), 4.17 (s, 1H), 6.44 (dd,
1H, J1 ) 3.7, J2 ) 6.2 Hz), 7.33 (m, 2H), 7.45 (m, 8H), 8.62 (s, 1H),
10.71 (s, 1H). 13C NMR (75 MHz, DMSO-d6): δ -3.91, -3.72, 2.04,
14.23, 17.63, 19.40, 25.68, 45.35, 45.35, 64.76, 86.35, 88.61, 91.96,
121.13, 127.11, 127.40, 129.26, 129.52, 141.75, 143.29, 150.27, 152.27,
(5′R)-(N 2-Isobutytryl-9-[5′-O-(4,4′-dimethoxytriphenyl)methyl-
2′-deoxy-3′-C, 5′-C-[(5′-C, 6′-C-methano)ethano]-â-D-ribofuranosyl]-
guanine (13). To a solution of 12 (76 mg, 202 µmol) in pyridine (0.7
mL) was added at room temperature (in four portions over 8 h)
DMTOTf (348 mg, 707 µmol). After another 2 h, the reaction mixture
was diluted with CH2Cl2 and the mixture extracted with saturated
NaHCO3. The combined organic layers were dried over MgSO4 and
concentrated. Flash chromatography (CH2Cl2/MeOH 15:1 f 10:1 +
0.02% Et3N) afforded 114 mg (83%) of 13 as a brown solid. Rf 0.62
(CH2Cl2/MeOH 10:1). 1H NMR (300 MHz, CD3OD): δ 0.50 (m, 1H),
0.92 (m, 1H), 1.24 (2s, 6H), 1.59 (m, 1H), 1.75 (m, 1H), 2.44 (dd, 1H,
J1 ) 6.6, J2 ) 13.6 Hz), 2.67 (s, 1H), 2.74 (m, 1H), 3.24 (m, 1H),
3.80, 3.81 (2s, 6H), 6.17 (d, 1H, J ) 5.1 Hz), 6.87 (m, 4H), 7.26 (m,
3H), 7.43 (m, 4H), 7.53 (m, 2H), 8.81 (s, 1H). 13C NMR (75 MHz,
CDCl3): δ 17.17, 19.06, 19.43, 23.27, 34.90, 40.65, 47.21, 55.24, 67.46,
85.98, 87.33, 86.87, 90.97, 113.00, 113.06, 121.53, 127.02, 127.76,
128.35, 130.63, 130.74, 136.88, 136.98, 146.58, 147.54, 148.06, 155.09,
154.16, 155.12, 175.15. IR (KBr): 3254, 2970, 1750, 1612, 1245 cm-1
.
158.46, 158.53, 180.41. IR (KBr): 3400, 3130, 1678, 1607, 1250 cm-1
.
MS (LSIMS): m/z (rel intensity) 757 (7), 417 (45), 341 (65), 297 (40),
251 (20), 225 (80), 196 (100), 168 (40). Data of 9: Rf 0.30 (hexane/
MS (LSIMS): m/z (rel intensity) 678 (9), 335 (12), 303 (100).
1
(5′R)-(N 2-Isobutyryl-9-[3′-O-(2-cyanoethoxy)(diisopropylamino)-
phosphino-5′-O-(4,4′-dimethoxytriphenyl)methyl-2′-deoxy-3′-C, 5′-
C-[(5′-C, 6′-C-methano)ethano]-â-D-ribofuranosyl]guanine (14). 2-Cy-
anoethyl N,N-diisopropylchlorophosphoramidite (77 µL, 0.33 mmol)
was added dropwise at room temperature to a solution of 13 (114 mg,
0.17 mmol) and N,N-diisopropylethylamine (114 µL, 0.17 mmol) in
CH3CN (0.4 mL) and THF (1.1 mL). After 5 h and 6 h, further portions
of 1 equiv of 2-cyanoethyl N,N-diisopropylchlorophosphoramidite were
added. After 10 h, 2-propanol was added, and the reaction mixture was
extracted with saturated NaHCO3. The combined organic layers were
dried over MgSO4 and concentrated. Flash chromatography (CH2Cl2/
acetone 3:1 + 0.02% Et3N) yielded 129 mg of 14 as a yellow oil.
Repeated precipitation from CH2Cl2 (0.3 mL) in hexane (100 mL) at 0
°C afforded 85 mg (58%) of 14 as a slightly brownish powder (3:2
EtOAc 2:1). H NMR (300 MHz, DMSO-d6): δ 0.04, 0.06 (2s, 6H),
0.11 (s, 9H), 0.77 (s, 9H), 0.80 (m, 1H), 0.97 (m, 1H), 1.10, 1.12 (2s,
6H), 1.56 (m, 1H), 1.78 (d, 1H, J ) 13.6 Hz), 2.31 (dd, 1H, J1 ) 4.0,
J2 ) 14.0 Hz), 2.74 (m, 1H), 2.91 (m, 2H), 4.51 (s, 1H), 6.34 (t, 1H,
J ) 7.0 Hz), 7.35 (m, 2H), 7.47 (m, 8H), 8.63 (s, 1H), 10.64 (s, 1H).
13C NMR (75 MHz, DMSO-d6): δ -3.88, -3.64, 1.99, 16.90, 17.68,
19.35, 19.44, 23.79, 25.75, 34.52, 39.63, 44.72, 65.38, 87.03, 84.14,
89.52, 120.85, 127.11, 127.41, 129.29, 129.57, 141.76, 143.60, 144.24,
150.36, 152.43, 154.42, 155.24, 175.03. IR (KBr): 2920, 1721, 1250
cm-1. MS (LSIMS): m/z (rel intensity) 757 (25), 417 (60), 341 (55),
225 (50), 196 (100), 168 (35).
(5′R)-(N2-Isobutyryl-9-[5′-O-(tert-butyldimethylsilyl)-2′-deoxy-3′-
C, 5′-C-[(5′-C, 6′-C-methano)ethano]-â-D-ribofuranosyl]guanine (11).
A solution of 9 (518 mg, 0.68 mmol) in DMSO (5 mL) was treated
with saturated NaNO2 in DMSO (15 mL) and stirred for 10 h at 60
°C. The reaction mixture was diluted with EtOAc at room temperature,
and the precipitate was filtered off. The recovered solution was
concentrated, and the residue was purified by flash chromatography
(CH2Cl2/MeOH 20:1 f 10:1) to afford 302 mg (90%) of 11 as a yellow
1
mixture of diastereoisomers). Rf 0.60 (CH2Cl2/acetone 3:1). H NMR
(400 MHz, CDCl3): δ 0.43 (m, 1H), 1.06 (m, 12H), 1.15 (m, 1H),
1.24 (m, 6H), 1.35 (m, 1H), 1.52 (m, 0.4H), 1.71 (m, 1H), 2.15 (dd,
0.6H, J1 ) 6.0, J2 ) 14.7 Hz), 2.60 (m, 4H), 3.18 (t, 1H, J ) 13.9
Hz), 3.42 (m, 4H), 3.79 (s, 6H), 6.03 (dd, 1H, J1 ) 5.2, J2 ) 12.1 Hz),
6.80 (m, 5H), 7.22 (m, 1H), 7.44 (m, 5H), 7.51 (m, 2H), 8.62 (s, 1H),
8.72 (s, 0.4H), 8.99 (s, 0.6H), 11.91 (s, 1H). 13C NMR (100 MHz,
1
oil. Rf 0.26 (CH2Cl2/MeOH 20:1). H NMR (300 MHz, CD3OD): δ
0.18, 0.24 (2s, 6H), 0.83 (m, 1H), 0.95 (s, 9H), 1.12 (m, 1H), 1.24,
9
J. AM. CHEM. SOC. VOL. 124, NO. 21, 2002 6001

A R T I C L E S
Renneberg and Leumann
CDCl3): δ 16.73, 16.94, 18.62, 18.72, 20.36, 20.44, 24.29, 24.95, 36.18,
38.28, 38.43, 42.71, 42.83, 42.94, 45.58, 55.08, 56.78, 56.85, 56.96,
57.03, 67.28, 67.40, 87.60, 88.36, 91.21, 92.17, 87.79, 87.85, 89.77,
89.83, 90.12, 112.74, 117.70, 122.34, 126.73, 127.54, 128.46, 130.55,
130.81, 137.01, 137.27, 137.78, 146.27, 146.54, 146.85, 155.56, 155.63,
158.45, 158.52, 178.26, 178.45. 31P NMR (161.9 MHz, CDCl3): δ
was suspended in concentrated NH3 solution (ca. 1 mL) and left
overnight at 55 °C, or for 63 h at 60-65 °C in those cases where the
universal solid support was used. The crude oligonucleotides were
purified by anion-exchange HPLC (Macherey-Nagel, Nucleogen DEAE
60/7) and desalted over SEP-PAK C-18 cartridges (Waters). Their purity
was controlled by RP-HPLC and estimated to be g95%. All oligode-
oxynucleotides were routinely analyzed by ESI mass spectrometry. The
corresponding MS data are given in Figure 3.
142.71, 142.88. IR (KBr): 3404, 2971, 1670, 1613, 1255 cm-1
.
X-ray Structures of 3 and 15. Data of 3. Crystal shape, rod; crystal
color, colorless; crystal size, 0.5 × 0.3 × 0.2 mm3; empirical formula,
C25H33N3O5Si (MW ) 483.63); crystal system, orthorhombic; space
group, P212121; unit cell dimensions (Å), a ) 6.2225(4), b ) 11.3686-
(6), c ) 35.120(2), R ) 90°, â ) 90°, γ ) 90°; volume, 2484.4(3) Å3;
reflections, 8000 (plus equivalents); angle range, 1.88 < θ < 25.92; Z,
4; density (calc.), 1.293 g/cm3; radiation used, Mo KR; wavelength,
0.71073 Å; linear absorption coefficient, 0.135 mm-1; temperature,
153(2) K; number of reflections measured, 16 272; number of
independent reflections, 4738; number of observed reflections, 3898;
R(int.) ) 0.0654; F(000), 1032. Data of 15. Crystal shape, rod; crystal
color, colorless; crystal size, 0.5 × 0.3 × 0.2 mm3; empirical formula,
C24H31N5O6 (MW ) 485.54); crystal system, orthorhombic; space
group, P212121; unit cell dimensions (Å), a ) 7.6651(5), b )
13.3047(7), c ) 23.7387(12), R ) 90°, â ) 90°, γ ) 90°; volume,
2420.9(2) Å3; reflections, 6776 (plus equivalents); angle range,
2.30 < θ < 25.92; Z, 4; density (calc.), 1.332 g/cm3; radiation used,
Mo KR; wavelength, 0.71073 Å; linear absorption coefficient, 0.097
mm-1; temperature, 153(2) K; number of reflections measured, 9936;
number of independent reflections, 4663; number of observed reflec-
tions, 3069; R(int.) ) 0.0382; F(000), 1032.
Oligonucleotide Synthesis and UV Melting Experiments. Syn-
theses of oligonucleotides were performed with solid-phase phosphor-
amidite methodology on the 1.0 or 1.3 µmol scale using a Pharmacia
LKB Gene Assembler Special instrument. CH3CN was distilled over
CaH2 and stored under activated molecular sieves (3 Å). The assembly
of tricyclo-DNA was performed according to the standard synthesis
cycles (trityl off mode) with the exception of a prolonged coupling
time (10 min) and an 11-fold instead of a 9-fold molar excess of
phosphoramidites. Because of solubility reasons, ClCH2CH2Cl was used
as the solvent for the tricyclodeoxyadenosine building block and a
mixture of CH3CN/THF ) 3:1 for the tricyclodeoxyguanosine building
block. In the coupling step, tetrazole (0.45 M in CH3CN) was replaced
by the more active 5-(benzylmercapto)-1H-tetrazole (0.25 M in CH3-
CN) or 5-(ethylmercapto)-1H-tetrazole (0.25 M in CH3CN). For
sequences 20-23, the universal solid support from CT-Gen (San Jose)
was used, and for all other oligonucleotides the standard CPG-solid
supports from Glen Research were used. The 5′-ends of oligotricy-
clodeoxynucleotides were equipped with a phosphate group by terminal
coupling with the phosphorylating reagent 2-[2-(4,4′-dimethoxytrityl-
oxy)ethylsulfonyl]ethyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphor-
amidite (Glen Research). For comparison, the natural oligonucleotides
were synthesized in the same way. After synthesis, the solid support
UV Melting Curves. UV melting curves were determined at 260
nm on a Varian Cary 3E spectrophotometer that was equipped with a
Peltier block using the Varian WinUV software. Complementary
oligodeoxynucleotides were mixed to 1:1 stoichiometry with a 2 µM
single strand oligonucleotide concentration. A heating-cooling-heating
cycle (0 f 90 °C) was applied with a temperature gradient of 0.5 °C/
min. Tm values were defined as the maximum of the first derivative of
the melting curve using the software package Origin 5.0. Thermody-
namic data for duplex formation were obtained as indicated in the text.
CD Spectra. CD spectra were recorded on a Jasco J-715 spectropo-
larimeter with a Jasco PFO-350S temperature controller. The temper-
ature was measured directly in the cell (path length 10 mm).
Molecular Modeling. Molecular modeling calculations were carried
out with the Amber force field as incorporated in the software package
Insight II/Discover 3 V98.0 of Molecular Simulations, Inc. on an SGI
Octane workstation. Only original Amber parameters and potentials
were used. No explicit H2O molecules and counterions were included.
A distance-dependent permittivity of ꢀ ) 4r was used instead as a
screening function. One to four nonbonded interactions were scaled
by 0.5. No cutoffs were applied. Structures were built on the basis of
the parameters of a B-DNA double helix. They were first minimized
until the energy gradient was below 0.05 kcal/mol Å. The structures
were then heated stepwise from 0 to 300 K over 20 ps (1 ps at 50 K,
1 ps at 100 K, 2 ps at 150 K, 2 ps at 200 K, 4 ps at 250 K, and 10 ps
at 300 K) and submitted to 200 ps of unrestrained molecular dynamics
at 300 K. The temperature was controlled by coupling to an external
heat bath. One-fs time steps were used in the numerical integration of
the equation of motion. For the analysis, coordinates and energy terms
were stored every 0.5 ps.
Acknowledgment. We thank the BENEFRI Small Molecule
Crystallography Service directed by Prof. Helen Stoeckli-Evans
for measuring the X-ray data sets. Financial support of this work
by the Swiss National Science Foundation (grant 20-63582.00)
is gratefully acknowledged.
Supporting Information Available: Experimental details on
the synthesis of the tricyclo-G nucleosides with O-6-phenethyl-
N-2-isobutyrylguanine as the base synthon (PDF). This material
is available free of charge via the Internet at http://pubs.acs.org.
JA025569+
9
6002 J. AM. CHEM. SOC. VOL. 124, NO. 21, 2002